WO1994007915A1 - New growth hormone releasing hormone receptor protein - Google Patents

New growth hormone releasing hormone receptor protein Download PDF

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Publication number
WO1994007915A1
WO1994007915A1 PCT/US1993/009084 US9309084W WO9407915A1 WO 1994007915 A1 WO1994007915 A1 WO 1994007915A1 US 9309084 W US9309084 W US 9309084W WO 9407915 A1 WO9407915 A1 WO 9407915A1
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sequence
protein
growth hormone
receptor
amino acid
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PCT/US1993/009084
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French (fr)
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Kelly E. Mayo
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Northwestern University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/60—Growth-hormone releasing factors (GH-RF) (Somatoliberin)

Definitions

  • This invention relates to purified naturally occurring proteins and to the corresponding protein produced by recombinant genetic techniques and more specifically to such proteins and genetic elements derived from a growth hormone releasing hormone receptor protein and to methods and compositions which employ the proteins and genetic elements.
  • GHRH neuroendocrine peptides growth hormone-releasing hormone
  • IGFs insulin-like growth factors
  • the IGF type I receptor which may function in both IGF-I and IGF-II signaling, is a protein tyrosine kinase, while the IGF type II receptor, which has high affinity for IGF-II, is a multifunctional protein that also serves as a receptor for mannose-6-phosphate.
  • Czech MP Signal Transmission by the Insulin-like Growth Factors. Cell 59:235-238 (1989), Humbel RE, Insulin-like Growth Factors I and II. Eur. J. Biochem. 190:445-462 (1990).
  • the growth hormone receptor is a member of the cytokine receptor family, and signal transduction by this receptor is not well understood. Kelly PA, et al.
  • GHRH also referred to as growth hormone-releasing factor, GRF
  • GRF growth hormone-releasing factor
  • GHRH belongs to a growing family of peptides which includes glucagon, vasoactive-intestinal peptide (VIP) , secretin, gastric inhibitory peptide (GIP) , peptide with histidine as N- terminus and isoleucine as C-terminus (PHI) , and pituitary adenylate cyclase activating peptide (PACAP) .
  • Bilezikjian L, Vale W Stimulation of Adenosine 3 '.5' Monophosphate Pro ⁇ duction by Growth Hormone-Releasing Factor and its Inhibition by Somatostatin in Anterior Pituitary Cells In Vitro. Endocrinology 113:1726-1731, Labrie F, et al. Growth Hormone-Releasing Factor Stimulates Adenylate Cyclase Activity in the Anterior Pituitary Gland. Life Sciences 33:2229-2233 (1983), Struthers R, et al., Nucleotide Regulation of Growth Hormone-Releasing Factor Binding to Rat Pituitary Receptors.
  • GHRH receptor in addition to playing a key role in normal growth regulation, might be important in disorders involving aberrant growth hormone secretion.
  • GTPase Inhibiting Mutations Activate the ⁇ chain of Gs and Stimulate Adenyl Cyclase in Human Pituitary Tumours, Nature 340:692-696 (1989), Vallar L, et al, Altered Gs and Adenylate Cyclase Activity in Human GH-Secreting Pituitary Adenomas. Nature 330:566-568 (1987), suggesting the possibility that the GHRH receptor itself could be a proto-oncogene subject to activation mutations in some pituitary tumors. Cooper JA, Oncogenes and Anti- Oncogenes. Curr. Opin. Cell. Biol. 2:286-295 (1990) .
  • GHRH receptor cDNA clones were identified from rat and human pituitary. These cDNAs encode proteins that have the expected features of G protein-coupled receptors, and are related to the recently identified secretin and VIP receptors.
  • the human cDNA when expressed in human kidney 293 cells, produces a protein that binds GHRH with high affinity and specificity and increases GHRH- dependent cAMP production by these transfected cells.
  • the highly related rat cDNA detects an mRNA that is specifically expressed in the rat anterior pituitary gland, the major target for GHRH action.
  • the present invention provides the cDNA sequence of the pituitary specific receptor for growth hormone releasing hormone as set out in Sequence Id. no. 1 and 2. More specifically, this invention provides a purified human growth hormone-releasing hormone receptor protein selected from the group consisting of a human growth hormone-releasing hormone receptor protein having an amino acid sequence which is at least 85% identical to the amino acid sequence of Sequence Id. No. 1 and fragments thereof comprising at least 10 consecutive amino acids of the sequence. Additionally, this invention provides a purified rat growth hormone- releasing hormone receptor protein selected from the group consisting of a rat growth hormone-releasing hormone receptor protein having an amino acid sequence which is at least 85% identical to the amino acid sequence of Sequence Id. No. 2 and fragments thereof comprising at least 10 consecutive amino acids of the sequence. Additionally, this invention provides a new growth hormone-releasing receptor protein using recombinant DNA molecules capable of expressing this new protein.
  • this invention provides a method to screen ligands for growth hormone releasing hormone activity. This screening technique could lead to new agonist and antagonist of growth hormone releasing hormone. Description of the Figures
  • Fig. IA shows the structure of the human pituitary GHRH receptor cDNAs and protein. Schematic maps of human GHRH receptor cDNA clones. A composite cDNA showing the location of the 1269-basepair open reading frame (shaded rectangle) is shown. Above this are the two human cDNA clones isolated, along with a partial restriction map of the HPR3 cDNA.
  • Fig. IB shows hydropathy plot of the human GHRH receptor protein. Seven strongly hydrophobic regions large enough to span the plasma membrane are numbered 1- 7.
  • Fig. IC shows DNA sequence and amino acid sequence of the human pituitary GHRH receptors. Amino acid 1 corresponds with nucleotides 52-54.
  • Fig. 2A shows the structure of the rat pituitary GHRH receptor cDNAs and protein. Schematic maps of rat GHRH receptor cDNA clones. A composite cDNA showing the location of the 1269 basepair open reading frame (shaded rectangle) is shown. Above this are the four rat cDNA clones isolated, along with a partial restriction map of the composite cDNA. RPR6 is the initial PCR-generated clone. The triangle indicates an insertion found in clone RPR13 relative to RPR11.
  • Fig. 2B shows hydropathy plot of the rat GHRH receptor protein. Seven strongly hydrophobic regions large enough to span the plasma membrane are numbered 1- 7.
  • Fig. 2C shows DNA sequence and amino acid sequence of the rat pituitary GHRH receptor. Amino acid 1 corresponds with nucleotides 28-30.
  • Fig. 3 shows a model for the generation of cDNA clones RPR11 and RPR13 via alternative RNA processing. The upper splicing pattern would generate the RPRll cDNA sequence, while the lower splicing pattern would generate the RPR13 cDNA sequence. Exon sequences are underlined and include amino acid sequence. Note that this model predicts the use of a non-consensus splice donor sequence from the alternative exon (bold characters) .
  • Fig. 4 shows binding of GHRH by the human GHRH receptor cDNA clone HPR3 in transfected human kidney 293 cells.
  • A binding of [l 25 l-Tyr 10 ] human GHRH (1-44)- amide to membranes from 293-HPR9 cells. The symbols are (•) total binding, ( ⁇ ) specific binding, (o) non-specific binding, which was determined by inclusion of excess unlabeled GHRH (1 ⁇ M) in parallel samples. Values shown are the means of duplicate samples, which varied by less than 5%.
  • B Scatchard analysis of the binding data. Regression analysis was performed using Data Desk 2.0 software. A Kd of 27 pM and 34 pM was determined in two independent experiments, one of which is shown here.
  • C Binding competition curve for GHRH in 293-HPR9 cells.
  • Fig. 5 shows stimulation of cAMP production by GHRH in human kidney 293 cells expressing the human GHRH receptor.
  • A stimulation of cAMP production by GHRH in 293-HPR9 cells. The results from two independent experiments are shown (• and ⁇ ) ; each was performed in triplicate and the symbols represent the mean plus and minus the standard error.
  • B Specificity of cAMP production by 293-HPR9 cells. Relative cAMP levels are shown as a function of the cell type (293-WT or 293-HPR9) and the inducing peptide, which was present at 1 ⁇ M. The dotted line shows basal cAMP levels.
  • r Fig. 6 shows a schematic diagram of the GHRH receptor as it spans a membrane. It is believed that the amino terminus is on the outside of the cell, while the carboxy terminus is inside of the cell.
  • compositions comprising recombinant proteins produced using generic sequences encoding GHRH receptors and fragments derived therefrom are provided, together with proteins isolated from natural sources, methods of using these compositions.
  • the GHRH receptor cDNAs used to produce the recombinant proteins were initially isolated from human and rat pituitary cDNA libraries using a two-step procedure. First, degenerate oligonucleotides encoding portions of membrane spanning domains 6 and 7 of receptors for calcitonin, Lin H, et al., Expression Cloning of an Adenylate Cvclase-Coupled Calcitonin Receptor.
  • nucleotides and amino acids are used in these figures and elsewhere in this specification. See Kiefer et aJL. WO 9203471 partially set out to provide general background information. A number of terms used in the art of genetic engineering and protein chemistry are used herein with the following defined meanings. Two nucleic acid fragments are "homologous" if they are capable of hybridizing to one another under hybridization conditions described in Maniatis et al. , op. cit.. pp.320-323.
  • homologous sequences can be identified that contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15- 25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash conditions for identification of clones from gene libraries (or other sources of genetic material) , as is well known in the art.
  • a DNA fragment is "derived from" a GHRH receptor encoding DNA sequence if it has the same or substantially the same basepair sequence as a region of the coding sequence for the entire GHRH receptor molecule.
  • substantially the same means, when referring to biological activities, that the activities are of the same type although they may differ in degree.
  • amino acid sequences substantially the same means that the molecules in question have similar biological properties and preferably have at least 85% identity in amino acid sequences. More preferably, the amino acid sequences are at least 90% identical. In other uses, substantially the same has its ordinary English language meaning.
  • a protein is "derived from" a GHRH receptor molecule if it has the same or substantially the same amino acid sequence as a region of the GHRH receptor molecule.
  • GHRH receptor both glycosylated and unglycosylated, or polypeptide derivatives thereof, may be used for producing antibodies, either monoclonal or polyclonal, specific to GHRH receptors.
  • polypeptide derivatives of these GHRH receptors is meant polypeptides differing in length from natural GHRH receptor and containing five or more amino acids from GHRH receptor in the same primary order as found in GHRH receptor as obtained from a natural source.
  • Derivatives include glycosylated forms and covalent conjugates with unrelated chemical moieties.
  • GHRH receptor-specific molecules include poly- peptides such as antibodies that are specific for the GHRH receptor polypeptide containing the naturally occurring GHRH receptor amino acid sequence.
  • specific binding polypeptide is intended polypeptides that bind with GHRH receptor and its derivatives and which have a measurably higher binding affinity for the target polypeptide, i.e., GHRH receptor and polypeptide derivatives of GHRH receptor, than for other polypeptides tested for binding. Higher affinity by a factor of 10 is preferred, more preferably a factor of 100. Binding affinity for antibodies refers to a single binding event (i.e., monovalent binding of an antibody molecule).
  • Specific binding by antibodies also means that binding takes place at the normal binding site of the molecule's antibody (at the end of the arms in the variable region) .
  • minor amino acid variations from the natural amino acid sequence of GHRH receptor are contemplated as being encompassed by the GHRH receptor; in particular, conservative amino acid replacements are contemplated.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • Antibodies specific for GHRH receptor are produced by immunizing an appropriate vertebrate host, e.g., rabbit, with purified GHRH receptor or polypeptide derivatives of GHRH receptor, by themselves or in conjunction with a conventional adjuvant. Usually, two or more immunizations will be involved, and blood or spleen will be harvested a few days after the last injection. For polyclonal antisera, the immunoglobulins can be precipitated, isolated and purified by a variety of standard techniques, including affinity purification using GHRH receptor attached to a solid surface, such as a gel or beads in an affinity column.
  • the splenocytes normally will be fused with an immortalized lymphocyte, e.g., a myeloid cell line, under selective conditions for hybridoma formation.
  • the hybridomas can then be cloned under limiting dilution conditions and their supernatants screened for antibodies having the desired specificity.
  • Techniques for producing antibodies are well known in the literature and are exemplififed by the publication Antibodies: A Laboratory Manual (a988) eds. Harlow and Lane, Cold Spring Harbor Laboratories Press, and U.S. Patent Nos. 4,381,292, 4,451,570, and 4,618,577.
  • GHRH receptor can be readily purified from pituitaries and its components, and from cells genetically modified to produce GHRH receptor or polypeptide derivatives thereof, by affinity chromatography using a monoclonal antibody specific for GHRH receptor.
  • GHRH receptor and polypeptide derivatives thereof can be purified by a variety of other widely known protein purification techniques (either alone or in combination) including immunoprecipitation, gel filtration, ion exchange chromatography, chromato- focusing, isoelectric focusing, selective precipitation, electrophoresis, and the like.
  • Isolation of nucleotide sequences encoding GHRH receptor involves creation of either a genomic library or preparation of a cDNA library from RNA isolated from cells expressing GHRH receptor. It will generally be preferable to create a cDNA library for isolation of GHRH receptor coding nucleotide sequences so as to avoid any possible problems arising from attempts to determine intron/exon borders. Genetic libraries can be made in either eukaryotc or prokaryotic host cells. Widely available cloning vectors such as plasmids, cosmids, phage, YACs and the like can be used to generate genetic libraries suitable for the isolation of nucleotide sequences encoding GHRH receptor or portions thereof.
  • Useful methods for screening genetic libraries for the presence of GHRH receptor nucleotide sequences include the preparation of oligonucleotide probes based on the N-terminus amino acid sequence information from purified GHRH receptor or purified internal fragments of purified GHRH receptor.
  • oligonucleotide sequences of about 17 base pairs or longer can be prepared by conventional in vitro synthesis techniques so as to correspond to portions of GHRH receptor for which the amino acid sequence has been determined by N-terminus analysis.
  • the resultant nucleic acid sequences can be subsequently labeled with radionuclides, enzymes, biotin, fluorescers, or the like, and used as probes for screening genetic libraries.
  • Additional methods of interest for isolating GHRH receptor encoding nucleic acid sequences include screening genetic libraries for the expression of GHRH receptor or fragments thereof by means of GHRH receptor specific antibodies, either polyclonal or monoclonal.
  • a particularly preferred technique involves the use of degenerate primers based on partial amino acid sequences of purified GHRH receptor or on sequences from known related molecules and the polymerase chain reaction (PCR) to amplify gene segments between the primers. The gene can then be isolated using a specific hybridization probe based on the amplified gene segment, which is then analyzed for appropriate expression of protein.
  • PCR polymerase chain reaction
  • Nucleotide sequences encoding GHRH receptor can be obtained from recombinant DNA molecules recovered from GHRH receptor genetic library isolates.
  • the nucleotide sequence encoding GHRH receptor can be obtained by sequencing the non-vector nucleotide sequences of these recombinant molecules.
  • Nucleotide sequence information can be obtained by employing widely used DNA sequencing protocols, such as Maxim and Gilbert sequencing, dideoxy nucleotide sequencing, and the like. Examples of suitable nucleotide sequencing protocols can be found in Berger and Kimmel, Me €hods in Enzymologv Vol. 52. Guide to Molecular Cloning Techniques, (1987) Academic Press.
  • Nucleotide sequence information from several recombinarr DNA isolates may be combined so as to provide th»> entire amino acid coding sequence of GHRH receptor, as well as the nucleotide sequences of introns within the GHRH receptor genes, upstream nucleotide sequences, and downstream nucleotide sequences.
  • Nucleotide sequences obtained from sequencing GHRH receptor specific genetic library isolates are subjected to analysis in order to identify regions of interest in the GHRH receptor genes. These regions of interest include open reading frames, introns, promoter sequences, termination sequences, and the like. Analysis of nucleotide sequence information is preferably performed by computer.
  • DNASISTM DNASISTM
  • Isolated nucleotide sequences encoding GHRH receptor can be used to produce purified GHRH receptor or fragments thereof by either recombinant DNA methodology or by in vitro polypeptide synthesis techniques.
  • purified and isolated is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • the term “purified” as used herein preferably means at least 95% by weight, more preferably at least 99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present) .
  • GHRH receptor by recombinant DNA techniques rather than by isolating GHRH receptor from natural sources is that equivalent quantities of GHRH receptor can be produced by using less starting material than would be required for isolating the binding protein from a natural source.
  • GHRH receptor by recombinant techniques also permits GHRH receptor to be isolated in the absence of some molecules normally present in cells that naturally produce GHRH receptor. Indeed, GHRH receptor compositions entirely free of any trace of human protein contaminants can readily be produced since the only human protein produced by the recombinant nonhuman host is the recombinant GHRH receptor. Potential viral agents from natural sources are also avoided. It is also apparent that recombinant DNA techniques can be used to produce polypeptide derivatives that are not found in nature, such as the variations described above.
  • GHRH receptor and polypeptide derivatives of GHRH receptor can be expressed by recombinant techniques when a DNA sequence encoding the relevant molecule is functionally inserted into a vector.
  • functionally inserted is meant in proper reading frame and orientation, as is well understood by those skilled in the art.
  • the preferred starting material is a cDNA library isolate encoding GHRH receptor than a genomic library isolate.
  • the GHRH receptor gene will be inserted downstream from a promoter and will be followed by a stop codon, although production as a hybrid protein followed by cleavage may be used, if desired.
  • host- cell-specific sequences improving the production yield of GHRH receptor and GHRH receptor polypeptide derivatives will be used and appropriate control sequences will be added to the expression vector, such as enhancer sequences, polyadenylation sequences, and ribosome binding sites.
  • the appropriate coding sequence can be expressed in a variety of different expression systems, or it can be inserted into the genome for transgenic expression.
  • Mammalian Expression Systems Mammalian Expression Systems
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a mammalian promoter will also contain an upstream promoter element, typically located within 100 to 200 bp upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation (Sambrook et ai. (1989) Expression of Cloned Genes in Mammalian Cells," in Molecular Cloning: A Laboratory Manual. 2nd ed.) .
  • Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP) , and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible) , and depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.
  • Enhancer element is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from t he promoter (Maniatis et al.. (1989) Molecular Biology of the Cell. 2nd ed.). Enhancer elements derived from viruses may be particularly useful, because they typically have a broader host range.
  • Examples include the SV40 early gene enhancer (Dijkema et al. (1985) EMBO J. 4:761) and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gormal et aJ (1982b) Proc. Natl. Acad. Sci. 79:6777) and from human cytomegalovirus (Boshart et al. (1985) Cell 41:521). Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion (Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237).
  • an inducer such as a hormone or metal ion
  • a DNA molecule may be expressed intracellularly in mammalian cells.
  • a promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
  • foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells.
  • the leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • the adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3 • to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • the 3' terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation (Birnstiel et al. (1985) Cell 41;349; Proudfoot and Whitelaw (1988) "Termination And 3' end processing of eukaryotic RNA.” In Transcription and splicing (ed. B.D. Hames and D.M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105). These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.
  • transcription terminator/polyadenylation signals include those derived from SV40 (Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning; A Laboratory Manual) .
  • introns also called intervening sequences
  • splicing signals also called spliced donor and acceptor sites
  • Introns are intervening noncoding sequences within a coding sequence that contain spliced donor and acceptor sites. They are removed by a process called "splicing" following polyadenylation of the primary transcript (Nevins (1983) Annu. Rev. Biochem. 52:441; Green (1986) Annu. Rev. Genet. 20:671; Padgett et al. (1986) Annu. Rev. Biochem. 55:1119; Krainer and Maniatis (1988) "RNA splicing.” In Transcription and splicing (ed. B.D. Hames and D.M. Glover)).
  • the above described components comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.
  • Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired.
  • Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria.
  • Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate.
  • plasmids containing the replication systems of papovaviruses such as SV40 (Gluzman (1981) Cell 23:175) or polyomavirus, replicate to extremely high copy number in the presence of the T antigen.
  • mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus.
  • the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a procaryotic host for cloning and amplification.
  • mammalian-bacteria shuttle vectors include pMT2 (Kaufman et a_A_ (1989) Mol. Cell. Biol.
  • foreign proteins can also be targeted to the membrane of a mammalian cell.
  • the cDNA expression construct includes an amino-terminal hydrophobic leader sequence, and one or more additional internal hydrophobic domains of sufficient size to span the cell membrane (typically -20 amino acids)
  • the resulting protein can be targeted to the cell membrane and retained there in a conformation dependent on the nature and characteristics of the internal hydrophobic domains.
  • a baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (3*) transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5* end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • a baculovirus promoter may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.
  • Sequences encoding genes abundantly transcribed at late times in the infection cycle provide particularly useful promoter sequences. Examples include sequences derived from the polyhedrin (Friesen et al. (1986) "The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler) ; E.P.O. Pub. Nos. 127,839 and 155,476) and plO (Vlak et al. (1988) J. Gen. Virol. 69:765) genes. A DNA molecular may be expressed intracellularly.
  • a promoter sequence may be directly linked with the DNA molecule, in which the case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
  • Fusion proteins provide an alternative to direct expression.
  • a DNA sequence encoding the N- terminal portion of an endogenous yeast protein, or other stable protein is fused to the 5' end of heterologous coding sequences.
  • this construct will provide a fusion of the two amino acid sequences.
  • the N-terminus of the polyhedrin gene may be linked at the 5' terminus of a foreign gene and expressed in yeast.
  • the DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See e.g., Luckow et al. (1988) Bio/technology 6:47.
  • foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects.
  • the leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene
  • leaders of non-baculovirus origin such as those derived from genes encoding human alpha-interferon (Maeda et al. (1985) Nature 315:592), human gastrin-releasing peptide (Lebacq-Verheyden et al. (1988) Molec. Cell. Biol. 8:3129), human IL-2 (Smith et a . (1985) Proc. Natl. Acad. Sci. USA 82:8404), mouse IL-3 (Miyajima et al. (1987) Gene 58:273), and human glucocerebrosidase (Martin et al. (1988) DNA 7:99) also provide for secretion in insects.
  • transcription termination sequences recognized by insects are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples include transcription termination sequences derived from the polyhedrin gene (Miller et al. (1988) Ann. Rev. Microbiol. 42:177). Prior to insertion of the foreign gene into the baculovirus genome, the above described components, comprising a promoter, leader (if desired) , coding sequence of interest, and transcription termination sequence, are typically put together into an intermediate transplacement construct.
  • a replicon such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria.
  • the replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host for cloning and amplification.
  • the promoter and transcription termination sequence of the construct will typically comprise a 2.5kb section of the baculovirus genome for integration of the foreign gene into the baculovirus genome by double crossover recombination events, producing a baculovirus expression vector (Miller et al. (1989) Bioessays 4:91).
  • the baculovirus expression vector is typically packaged into an infectious recombinant baculovirus.
  • selectable markers are, such as antibiotic resistance genes, are generally not used. Selection is typically by visual inspection for occlusion bodies. Examples are given elsewhere in this specification of the use of selectable markers.
  • Recombinant baculovirus expression vectors have been developed for infection into several insect cells.
  • recombinant baculoviruses have been developed for inter alia: A ⁇ des aegypti, A tographa californica, Bombyx mori, Drosophila melanogaster, Heliothis zea, Spodoptera frugiperda, and Tric oplusiain (P.C.T. WO 89/046699; Carbonell et aj ⁇ (1985) J. Virol. 56:153: Smith et al*. (1983) Mol. Cell. Biol. 3:2156; Wright (1986) Nature 321:718; See generally, Fraser et S-L. (1989) In Vjtfp ⁇ *U . pgv ⁇ Bj ⁇ >; t 25:225).
  • Methods of introducing exogenous DNA into insect hosts are well-known in the art, and typically include either the transfection of host insect cells with DNA or the infection of insect cells or live insects, usually larvae, with virus. Transfection procedures are based on the calcium phosphate procedure originally developed for mammalian cells (Graham ej£ al. (1973) Virology 52:456) . DNA transfection and viral infection procedures usually vary with the insect genus to be transformed. See e.g. Autograph (Carstens et al. (1980) Virology 101:311), Heliothis (virescens) (P.C.T. Pub. No. W088/02030) , Spodoptera (Kang (1988) "Baculovirus Vectors for Expression of Foreign Genes," in Advances in Virus Research, vol. 35).
  • foreign proteins can also be targeted to the membrane of an insect cell.
  • the cDNA expression construct includes an amino-terminal hydrophobic leader sequence, and one or more additiona. internal hydrophobic domains of sufficient size to spa.-. the cell membrane (typically -20 amino acids)
  • the resulting protein can be targeted to the cell membrane and retained there in a conformation dependent on the nature and characteristics of the internal hydrophobic domains.
  • a bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g., structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • a bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA * polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.
  • Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.
  • positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence.
  • An example of a gene activator protein is the catabolite activator protein (CAP) , which helps initiate transcription of the lac operon in Escherichia coli (E. coli) (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
  • CAP catabolite activator protein
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.
  • Example include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1977) Nature 198:1056). and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al*. (1981) Nucl. Acids Res. 9:731: U.S. Patent No. 4,738,921; E.P.O. Pub. Nos. 36,776 and 121,775).
  • trp tryptophan
  • synthetic promoters which do not occur in nature also function as bacterial promoters.
  • transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter (U.S. Patent No. 4,551,433).
  • the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor (Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21).
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
  • a naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to product high levels of expression of some genes in prokaryotes.
  • the bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1985) Proc. Natl. Acad. Sci. 82:1074).
  • a hybrid promoter can also be comprised of a bacteriophage promoter and an E_j_ coli operator region (E.P.O. Pub. No. 267,851) .
  • an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes.
  • the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (Shine et al. (1975) Nature 254:34).
  • SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3 • and of E . coli 16S rRNA (Steitz et al.
  • a DNA molecule may be expressed intracellularly.
  • a promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N- terminus will always be a methionine, which is encoded by the ATG start codon.
  • methionine at the N- terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo or in vitro incubation with a bacterial methionine N- terminal peptidase (E.P.O. Pub. No. 219,237). Fusion proteins provide an alternative to direct expression.
  • a DNA sequence encoding the N- terminal portion of an endogenous bacterial protein, or other stable protein is fused to the 5' end of heterologous coding sequences.
  • this construct will provide a fusion of the two amino acid sequences.
  • the bacteriophage lambda cell gene can be linked at the 5' terminus of a foreign gene and expressed in bacteria.
  • the resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave, the bacteriophage protein from the foreign gene (Nagai et al. (1984) Nature 309:810). Fusion proteins can also be made with sequences from the lacZ (Jia et aJ .
  • the DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.
  • a ubiquitin fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein.
  • a processing enzyme e.g. ubiquitin specific processing-protease
  • foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria (U.S. Patent No.
  • the signal sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell(gram-negative bacteria).
  • processing sites which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.
  • DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E_j. coli outer membrane protein gene (ompA) (Masui et al. (1983) , in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212).
  • the signal sequence of the alpha- amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B___ subtilis (Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; E.P.O. Pub. No. 244,042) .
  • transcription termination sequences recognized by bacteria are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forminq stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E ⁇ . coli as well as other biosynthetic genes.
  • Expression constructs are often maintains ⁇ in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria.
  • a replicon such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria.
  • the replicon will have a replication system, thus allowing it to be maintained in a procaryotic host either for expression or for cloning and amplification.
  • a replicon may be either. a high or low copy number plasmid.
  • a high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and typically about 10 to about 150.
  • a host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.
  • the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors typically contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (E.P.O. Pub. No. 127,328).
  • Integrating vectors may also be comprised of bacteriophage or transposon sequences.
  • extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.
  • Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin) , and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469).
  • Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
  • Transformation vectors are typically comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.
  • Expression and transformation vectors have been developed for transformation into many bacteria.
  • expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis (Palva et al.. (1982) Proc. Natl. Acad. Sci. USA 79:5582; E.P.O. Pub. Nos. 36,259 and 63,953; P.C.T. WO 84/04541), Escherichia coli (Shimatake et al. (1981) Nature 292:128; Aman et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol.
  • Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and typically include either the transformation of bacteria treated with CaCl 2 or other agents, such as divalent cations and DMSO.
  • DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See e.g. , (Masson et a . (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; E.P.O. Pub. Nos. 36,259 and 63,953; P.C.T. WO 84/04541, Bacillus) , (Miller et al.
  • the resulting protein can be targeted to the cell membrane and retained there in a conformation dependent on the nature and characteristics of the internal hydrophobic domains. (Wickner W.T. and Lodish H.F., Multiple Mechanisms of Protein Insertion into and Across Membranes. Science 300:400-407 (1985)). (Hereby incorporated by reference) .
  • a yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a yeast expression system is particularly important in the present application for the expression of the binding protein which is expected to be extracellular. See Fig. 6.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site (the "TATA Box") and a transcription initiation site.
  • a yeast promoter may also have a second domain called an upstream activator sequence (UAS) , which, if present, is usually distal to the structural gene.
  • the UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.
  • Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydorgenase (ADH) (E.P.O. Pub No. 284044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH) , hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (E.P.O. Pub. No. 329203) .
  • ADH alcohol dehydorgenase
  • GAP or GAPDH glyceraldehyde-3-phosphate-dehydrogenase
  • hexokinase phosphofructokinase
  • 3-phosphoglycerate mutase
  • the yeast PHQ5 gene encoding acid phosphatase, also provides useful promoter sequences (Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1).
  • synthetic promoters which do not occur in nature also function as yeast promoters.
  • UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.
  • hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Patent Nos. 4,876,197 and 4,880,734).
  • Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2.
  • a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yease RNA polymerase and initiate transcription. Examples of such promoters include inter alia. (Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al ⁇ . (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96: 119; Hollenberg et al.
  • a DNA molecle may be expressed intracellularly in yeast.
  • a promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant progein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
  • Fusion proteins provide an alternative to direct expression.
  • a DNA sequence encoding the N- terminal portion of an ' endogenous yeast protein, or oth. r stable protein is fused to the 5* end of heterologous coding sequences.
  • this construct wil. provide a fusion of the two amino acid sequences.
  • the yeast or human superoxide dismutase (SOD, gene can be linked at the 5' terminus of a foreign gene and expressed in yeast.
  • the DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See e.g., E.P.O. Pub. No. 196056.
  • Another example is a ubiquitin fusion protein.
  • Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein.
  • a processing enzyme e.g. ubiquitin-specific processing protease
  • foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein.
  • there are processing sites encoded between the leader fragment and the foreign gene tht can be cleaved either in vivo or in vitro.
  • the leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (E.P.O. Pub. No. 13873; J.P.O. Pub. No. 62,096,086) and the A-factor gene (U.S. Patent No. 4,588,684).
  • yeast invertase gene E.P.O. Pub. No. 13873; J.P.O. Pub. No. 62,096,086
  • A-factor gene U.S. Patent No. 4,588,684
  • leaders of non-yeast origin such as an interferon leader, exist that also provide for secretion in yeast (E.P.O. Pub. No. 60057).
  • a preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre" signal sequence, and a "pro” region.
  • the types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (typically about 25 to about 50 amino acid residues) (U.S. Patent Nos. 4,546,083 and 4,870,008; E.P.O. Pub. No. 324274).
  • Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast slphafactor. (See e.g., P.C.T. WO 89/02463) .
  • transcription termination sequences recognized by yeast are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.
  • expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as yeast or bacteria.
  • the replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a procaryotic host for cloning and amplification.
  • yeast-bacteria shuttle vectors include YEp24 (Botstein et al. (1979) Gene 8:17- 24), pCl/1 (Brake et al ⁇ _ (1984) Proc Natl.
  • a replicon may be either a h igh or low copy number plasmid.
  • a high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and typically about 10 to about 150.
  • a host containing a high copy number plasmit will preferably have at least about 10, and more preferably at least about 20. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See e.g. Brake et al. , supra.
  • the expression constructs can be integrated into the yeast genome with an integrating . vector.
  • Integrating vectors typically contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome (Orr-Weaver et al. (1983) Methods in Enzymol. 101:228- 245) .
  • An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al. , supra.
  • One or more expression construct may integrate, possibly affecting levels of recombinant protein produced (Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750).
  • the chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.
  • extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.
  • Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2. HIS4. LEU2. TRP1. and ALG7. and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively.
  • a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal.
  • the presence of CUP1 allows yeast to grow in the presence of copper ions (Butt et al. (1987) Microbiol. Rev. 51:351).
  • transformation vectors are typically comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
  • Expression and transformation vectors either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts.
  • expression vectors have been developed for, inter alia, the following yeasts: Candida albicans
  • yeast cells treated with alkali cations Transformation procedures usually vary with the yeast species to be transformed. See e.g., (Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze ejt al. (1985) J. Basic Microbiol. 25:141; Candida ) ; (Gleeson et al. (1986) J. Gen.
  • the cDNA expression construct includes an amino-terminal hydrophobic leader sequence, and one or more additional internal hydrophobic domains of sufficient size to spar, the cell membrane (typically -20 amino acids)
  • the resulting protein can be targeted to the cell membrane and retained there in a conformation dependent on the nature and characteristics of the internal hydrophobic domains.
  • the genetic material of the invention can itself . used in numerous assays as probes for genetic ateria. present in naturally occurring materials.
  • the analytt- can be a nucleotide sequence which hybridizes with a probe comprising a sequence of (usually) at least about 16 consecutive nucleotides, usually 30 to 200 nucleotides, up to substantially the full sequence of the sequences shown above (cDNA sequences) .
  • the analyte can be RNA or cDNA.
  • the sample is typically as described in the previous section.
  • a positive result is generally characterized as identifying a genetic material comprising a sequence at least about 70% identical to a sequence of at least 12 consecutive nucleotides of the sequences given herein, usually at least about 80% homologous to at least about 60 consecutive nucleotides within the sequences, and may comprise a sequence substantially identical to the full-length sequences.
  • the probe may contain a detectable label. Probes that are particularly useful for detecting RNA or DNA for receptor proteins are based on conserved regions of these proteins, particularly from amino acids 131 and amino acids 380 (See Fig. 6) showing the membrane spanning domain.
  • PCR technique One method for amplification of target nucleic acids, for later analysis by hybridization assays, is known as the polymerase chain reaction or PCR technique.
  • the PCR technique can be applied to detecting GHRH receptor of the invention in suspected samples using oligonucleotide primers spaced apart from each other and based on the genetic sequence set forth herein.
  • the primers are complementary to opposite strands of a double stranded DNA molecule and are typically separated by from about 50 to 450 nt or more (usually not more than 2000 nt) .
  • This method entails preparing the specific oligonucleotide primers and then repeated cycles of target DNA denaturation, primer binding, and extension with a DNA polymerase to obtain DNA fragments of the expected length based on the primer spacing. Extension products generated from one primer serve as additional target sequences for the other primer.
  • the degree of amplification of a target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2n where n is the number of cycles. Given that the average efficiency per cycle ranges from about 65% to 85%, 25 cycles produce from 0.3 to 4.8 million copies of the target sequence.
  • the PCR method is described in a number of publications, including Saiki et al.. Science (1985) 2309:1350-1354; Saiki et al..
  • the invention includes a specific diagnostic method for determination of GHRH receptor, based on selective amplification of GHRH receptor-encoding RNA fragments from tissue.
  • This method employs using reverse transcriptase to make a cDNA sequence and then amplifying this sequence with receptor specific primers.
  • These "primer fragments,” which form one aspect of the invention, are prepared from GHRH receptor fragments such as described above. The method follows the process for amplifying selected nucleic acid sequences as disclosed in U.S. Patent No. 4,683,202, as discussed above.
  • the present cDNA sequences provide proteins that can be used to screen altered ligands as agonists or antagonists to growth hormone releasing hormone. These altered ligands could be used to treat diseases associated with growth hormone releasing hormone. Similarly, with the cDNA sequence known, a protein could be designed to bind to growth hormone releasing hormone. This new receptor administered in a pharmaceutically acceptable carrier may be used to control growth rates in domestic animals. Additionally, human growth disorders can be assessed using either an antibody to the GHRH receptor or a nucleic acid probe to the receptor.
  • RPR6 encoded a peptide with strong homology to the targeted regions of the calcitonin, PTH and secretin receptors.
  • RPR6 was used to screen human and rat pituitary cDNA libraries, and a human cDNA clone of 1617 bases (HPR3) was identified that contained an open reading frame encoding a 47 kilodalton protein of 423 amino acids (Figura IA and Sequence Id. No. 1) .
  • the EC50 for competition of radioligand binding by hGHRH was found to be -50 pM. These values are in reasonable agreement witht he reported dissociation constants for binding of hGHRH (1-44) -amide or the related agonist (His 1 , Nle 27 ) hGHRH (1-32) -amide to rat pituitary membranes, which range from 41 pM to 680 pM, Abribat T. , et al. Characterization of ( Hi-Tyr ⁇ ) Human Growth Hormone Releasing Factor (1-44) Amide Binding to Rat Pituitary: Evidence for High and Low Affinity Classes of Sites. Brain Research 528:291-299 (1990), Seifert H, et al.
  • GHRH treatment resulted in a large increase in cAMP levels in 293-HPR9 cells, but had very little effect on cAMP levels in the control 293-WT cells.
  • Secretin had no effect on cAMP levels in the transfected 293-HPR9 cells, but VIP did increase cAMP to about 5% the level obtained with GHRH ( Figure 5B) , consistent with the ability of VIP to bind to the receptor with low affinity.
  • the activation of the intracellular signaling system upon the binding of a ligand to GHRH is shown by increased production of cyclic AMP or protein kinase C.
  • the tissue distribution of GHRH receptor mRNA expression was examined in the rat using the RPR64 cDNA as a hybridization probe.
  • the pituitary expression of the rat GHRH receptor was also analyzed by in situ hybridization using 35 s- labeled RNA probes. GHRH mRNA was not detected in the liver, but was detected in the pituitary using an antisense RNA probe. No hybridization was observed when a control sense-strand GHRH receptor probe was used.
  • the Pituitary-Specific Transcription Factor GHF-1 is a Homeobox-Containing Protein.. Cell 55:505-518 (1988) , Ingraham, et al..
  • oligonucleotide primers to membrane spanning domains 6 (5'-ACCCTC[CGA]TNCTG[GA]T[CG] CCGCTC[TC]T[TC]GG-3* (Sequence Id. No. 4) and 7 (5'-ACCCTC[CGA]TNCTG[GA]T[CG] CCGCTC[TC]T[TC]GG-3* (Sequence Id. No. 4) and 7 (5'-ACCCTC[CGA]TNCTG[GA]T[CG] CCGCTC[TC]T[TC]GG-3* (Sequence Id. No. 4) and 7 (5'-
  • TGCAC[CT]TCA[CT] [CG] [AG]TTG[AC] [AG] [AG]AA[AG]CA[AG]TA-3 ' (Sequence Id. No. 5) of the secretin, calcitonin, and PTH receptors were used to amplify cDNA generated from male rat pituitaries.
  • N is G, A, T or C.
  • Amplification was carried out for 30 cycles using an annealing temperature of 55°C on a Perkin Elmer Cetus thermocycler and Taq DNA polymerase (Perkin Elmer Cetus, Norwalt, CT) .
  • the product was gel-purified and cloned into pGEM3Z (Promega, Madison, WI) for DNA sequence analysis.
  • RPR6 was subsequently used to screen human and rat cDNA libraries, which were from Clonetech (Palo Alto, CA) .
  • the human pituitary library was constructed from a growth hormone-producing adenoma from a female patient in ⁇ bluemid, the rat pituitary library was constructed from adult males in ⁇ gtlO. Screening and plaque purification was performed using standard methods, and inserts were subcloned into pGEM3Z or pGEM7Z (Promega, Madison, WI) for ruther analysis.
  • cDNA clones were sequenced on both strands using modified T7 polymerase (United States Biochemicals, Cleveland OH) and dideoxynucleotides.
  • DNA sequence manipulations were performed using GeneWorks 2.1 software from Intelligenetics (Mountain View, CA) , Hydrophobicity was analyzed using the algorithm of Kyte and Doolittle, Kyte and Doolittle, A Simple Model for Displaying the Hydropathic Character of a Protein. J. Mol. Biol. 157:105-132 (1982), with an analysis window of 11 amino acids. Amino acid sequence alignment was performed using GeneWorks 2.1 software and was subsequently manually modified to maximize alignment of the cysteine residues.
  • Human cDNA clone HPR3 was subcloned into the eukaryotic expression vector pcDNA-1 (Invitrogen, San Diego, CA) using lipofectin reagent (Gibco-BRL,
  • control 293-WT cells or transfected 293- HPR9 cells were washed with phosphate-buffered saline and homogenized (20 strokes in a TEFLON-glass unit) on ice in 50 mM Tris-HCl pH 7.4, 5mM MgCL 2 , 2 mM EGTA, and 0.1 ⁇ M PMSF.
  • the homogenate was centrifuged for 5 minutes at lOOXg to remove larger material, and the supernatant was re-centrifuged for 10 minutes at 4000Xg.
  • the membrane pellet was resuspended in binding buffer: 25 mM Hepes LH 7.4, 50 mM NaCl, 5 mM MgCl 2 , 1 mM EGTA, 0.1 mM PMSF, o . mg/ml leupeptin, 1 mg/ml bacitractin and 0.1% bovine serum albumin. Binding reactions were performed at 23 ' for 60 minutes in a volume of 0.5 ml with -50 ⁇ g me br ⁇ protein. For competition experiments, ( 125 l-Tyr 10 ) - - GHRH (1-44) -amide (Amersham, Arlington Heights, IL) * present at -75 pM.
  • Binding reactions were terminated by centrifugation in a microfuge for 5 minutes in the cold. The supernatant was aspirated, and the tip of the tube containing the pellet was cut off and counted in a gamma counter. Total binding was generally 30-45% of input, while non-specific binding was 8-11% of input.
  • cAMP radioimmunoassays were performed as described, Nikaido S and Takahashi J, Twenty-four Hour Oscillations of cAMP in Chick Pineal Cells: Role of cAMP in the Acute and Circadian Regulation of Melatonin Production. Neuron 3:609-619 (1989). All peptides were the human sequences, and they were obtained from Peptides International (Louisville, KY) or Peninsula Laboratories (Belmont, CA) .
  • RNA was prepared from the indicated tissues by homgenization in guanidine isothiocynate and centrifugation through cesium chloride, and polyadenylated RNA was selected by chromatography using oligo(dT) cellulose. Approximately 10 ⁇ g of each RNA was separated by electrophoresis on denaturing 1% agarose/formaldehyde gels. RNA was transferred to a nylon membrane (ICN, Irvine, CA) , covalently attached by UV cross-linking, and detected by hybridization to the insert from RPR-64 that had been labeled with 32 P-dCTP using random hexamer primers and the Klenow fragment of E. coli DNA polymerase.
  • Hybridization was performed in 50% formamide, 5X SSPE, 2X Denhardt's reagent, 10% dextran sulfate, 0.1% SDS, and 100 ⁇ g/ml salmon sperm DNA.
  • the membranes were subsequently washed in 0.1X SSC at 65°C and exposed to Kodak XAR-5 film. After removal of probe in 50% formamide at 65°C, the membranes were re- hybidized to cDNA clone CHO-B, Harpold MM, et al. Production of mRNA in Chinese Hamster Cells:
  • ovarian sections were fixed in 5% paraformaldehyde (pH 7.5) for 5 min, washed in 2X SSC followed by 0.1 M triethanolamine (pH 8.0) and incubated in 0.25% acetic anhydride in 0.1 M tri- ethanolamine (pH 8.0) for 10 min. Sections were dehydrated using ethanol and vacuum dried. Antiserse and sense ( 35 S)UTP-labeled RNA probes were synthesized using T7 or SP6 polymerase. The RNA probe (2X10 7 cpm/ml in hybridization buffer: 50% formamide, 5X SSPE, 2X Denhardt's reagent, 10% dextran sulfate, 0.1% SDS, and
  • ADDRESSEE TILTON, FALLON, LUNGMUS & CHESTNUT
  • B STREET: 100 S. Wac er Drive, Suite 960
  • NAME FENTRESS, SUSAN B.
  • CTCTTCGTGG CCATCACCAT CCTGGTTGCT CTCAGGAGGC TCCACTGCCC CCGGAACTAC 540
  • GCCGTCTACC TGAACTGCCT CCTGGCCTCC ACCTCCCCCA GCTCAAGGAG AGCCTTCTGG "30
  • ATCTTCAACT TCCTGCCAGA CAATGCTGGC CTGGGCATCC GCCTCCCCCT GGAGCTGGGA . . - 3
  • TAGGCTGCCT CATCACGCCA CTGGAGTCCA CACTTGAATT TGGGCAGCTA CCACGGGTCT .
  • CTCCCTGTGC TATGCACTGG TACGTGGGTG GGCTGCAAAC TGGCTTTTGA GGACACTGCG 840

Abstract

A purified protein selected from the group consisting of growth hormone-releasing hormone receptor protein having an amino acid sequence which is at least 85 % identical to the amino acid sequence of Sequence Id. No. 1 or 2 and fragments thereof comprising at least 10 consecutive amino acids of the sequence. Recombinant DNA molecules encoding the binding proteins and subsequences thereof are also described along with recombinant microorganisms and cell lines containing the DNA molecules and methods for preparing the proteins by growing the recombinant hosts containing the relevant DNA molecules. Antibodies to the protein which are useful in various diagnostic applications, are also described. Animal or yeast cells expressly GHRH could be used to screen for agonist or antagonist of GHRH.

Description

NEW GROWTH HORMONE RELEASING HORMONE RECEPTOR PROTEIN
Field of the Invention
This invention relates to purified naturally occurring proteins and to the corresponding protein produced by recombinant genetic techniques and more specifically to such proteins and genetic elements derived from a growth hormone releasing hormone receptor protein and to methods and compositions which employ the proteins and genetic elements.
Background of the Invention The growth of vertebrate organisms is regulated in part by a complex cascade of hormones, including the neuroendocrine peptides growth hormone-releasing hormone (GHRH) and somatostatin, the pituitary protein growth hormone, and the insulin-like growth factors (IGFs) or somatomedins. Martin JB, Brain Mechanisms for
Integration of Growth Hormone Secretion. Physiologist 22:23-29 (1979), Hall K, Sara, Growth and Somatomedins. Vitamins and Hormones 40:175-233 (1983), Muller EE, Cocchi D, Locatelli V (eds) , Advances in Growth Hormone and Growth Factor Research. Springer Veriag, Berlin, 477 pp. (1989) . The actions of these hormones are mediated by specific cell-surface receptors, all of which have been identified and cloned with the exception of the receptor for GHRH. Ulrich A, et al., Insulin-like Growth Factor I Receptor Primary Structure: Comparison With Insulin Receptor Suggests Structural Determinants that Define Functional Specificity. EMBO Journal 5:2503-2512 (1986), Morgan D, et al., Insulin-like Growth Factor II Receptor as a Multifunctional Binding Protein. Nature 329:301-307 (1987), Leung D, et al., Growth Hormone Receptor and Serum Binding Protein; Purification. Cloning and Expression. Nature 330:537-543 (1987), Yamada Y, et al. , Cloning and Functional Characterization of a Family of Human and Mouse Somatostatin Receptors Expressed in Brain, Gastrointestinal Tract, and Kidney. Proc. Natl. Acad. Sci. USA 89:251-255 (1992). These receptors have diverse structures and signaling mechanisms. The IGF type I receptor, which may function in both IGF-I and IGF-II signaling, is a protein tyrosine kinase, while the IGF type II receptor, which has high affinity for IGF-II, is a multifunctional protein that also serves as a receptor for mannose-6-phosphate. Czech MP, Signal Transmission by the Insulin-like Growth Factors. Cell 59:235-238 (1989), Humbel RE, Insulin-like Growth Factors I and II. Eur. J. Biochem. 190:445-462 (1990). The growth hormone receptor is a member of the cytokine receptor family, and signal transduction by this receptor is not well understood. Kelly PA, et al. , The Prolactin/Growth Hormone Receptor Family. Endocrine Rev. 3:235-251 (1991). Defects in the growth hormone receptor in patients with Laron dwarfism demonstrate its important role in growth. Godowski PJ, et al., Characterization of the Human Growth Hormone Receptor Gene and Demonstration of a Partial Gene Deletion in Two Patients with Laron- type Dwarfism. Proc. Natl. Acad. Sci. USA 86:8083-8087 (1989) . Lastly, two recently identified somatostatin receptors, Yamada Y, et al., Cloning and Functional Characterization of a Family of Human and Mouse Somatostatin Receptors Expressed in Brain. Gastrointestinal Tract, and Kidney. Proc. Natl. Acad. Sci. USA 89:251-255 (1992), have the seven membrane- spanning domains characteristic of receptors that are coupled to intracellular signaling pathways by G proteins. Ross EM, Signal sorting and Amplification Through G Protein-coupled Receptors. Neuron 3:141-152 (1989) .
GHRH (also referred to as growth hormone-releasing factor, GRF) is a peptide hormone (42-44 amino acids in various species) that is synthesized in the hypothalamus and stimulates the secretion of growth hormone from pituitary somatotropes. Frohman L, Jansson J-O, Growth Hormone-Releasing Hormone. Endocrine Rev. 7:223-253 (1986) , Gelato MC, Merria CR, Growth Hormone Releasing Hormone. Ann. Rev. Physiol. 48:569-591 (1986), Guillemin R, et al.. Growth Hormone-Releasing Factor from a Human Pancreatic Tumor that Caused Acromegaly. Science 218:585- 587 (1982), Rivier J, et al., Characterization of a Growth Hormone-Releasing Factor From a Human Pancreatic Islet Tumour. Nature 300:276-278 (1982). GHRH belongs to a growing family of peptides which includes glucagon, vasoactive-intestinal peptide (VIP) , secretin, gastric inhibitory peptide (GIP) , peptide with histidine as N- terminus and isoleucine as C-terminus (PHI) , and pituitary adenylate cyclase activating peptide (PACAP) . Rivier J, et al., Characterization of a Growth Hormone- Releasing Factor From a Human Pancreatic Islet Tumour. Nature 300:276-278 (1982), Bell GI, The Glucagon Superfa ilv: Precursor Structure and Gene Organization. Peptides 7 (Suppl) :27-36 (1986), Miyata A, et al..
Isolation of a Novel 38 Residue Hvpothalamic Polypeptide Which Stimulates Adenylate Cyclase in Pituitary Cells. Biochem. Biophys Res. Comm. 164:567-574 (1989). Specific, high affinity binding sites for GHRH have been measured on pituitary membranes using several different iodinated GHRH analogues, Abribat T, et al..
Figure imgf000005_0001
Releasing Factor (1-44) Amide Binding to Rat Pituitary: Evidence for High and Low Affinity Classes of Sites. Brain Research 528:291-299, Seifert H, et al.. Binding Sites for Growth Hormone Releasing Factor on Rat Anterior Pituitary Cells. Nature 313:487-489 (1985), Velicelebi G, et al., Specific Binding of Synthetic Human Pancreatic Growth Hormone Releasing Factor (1-40-OH) to Bovine Anterior Pituitaries. Biochem. Biophys, Res. Comm.
126:33-39 (1985), and GHRH binding proteins ranging in size from 26-72 kilodaltons have been identified by chemical cross-linking with anterior pituitary cells. Velicelebi G, et al., Covalent Cross-linking of Growth Hormone-Releasing Factor to Pituitary Receptors.
Endocrinology 118:1278-1283 (1986), Zysk J, et al.. Cross-linking of a Growth Hormone Releasing-Factor Binding Protein in Anterior Pituitary Cells. J. Biol. Chem. 261:16781-16784 (1986). GHRH binding to pituitary cells results in the activation of adenylate cyclase, and guanine nucleotides inhibit GHRH binding, suggesting that Gs is an intermediate GHRH action. Bilezikjian L, Vale W, Stimulation of Adenosine 3 '.5' Monophosphate Pro¬ duction by Growth Hormone-Releasing Factor and its Inhibition by Somatostatin in Anterior Pituitary Cells In Vitro. Endocrinology 113:1726-1731, Labrie F, et al. Growth Hormone-Releasing Factor Stimulates Adenylate Cyclase Activity in the Anterior Pituitary Gland. Life Sciences 33:2229-2233 (1983), Struthers R, et al., Nucleotide Regulation of Growth Hormone-Releasing Factor Binding to Rat Pituitary Receptors. Endocrinology 124:24- 29. Consistent with the notion that cAMP is an important second messenger for GHRH signaling, both GHRH and cAMP increase pituitary growth hormone gene expression, induce the expression of the proto-oncogene c-fos, and stimulate the proliferation of pituitary somatotropic cells. Barinaga M, et al., Transcriptional Regulation of Growth Hormone Gene Expression by Growth Hormone-Releasing Factor. Nature 306:84-85 (1983), Gick, GG, et al.. Growth Hormone-Releasing Factor Regulates Growth Hormone mRNA in Primary Cultures of Rat Pituitary Cells. Proc. Natl. Acad. Sci. USA 81:1553-1555 (1984), Billestrup N, et al.. Growth Hormone-Releasing Factor Induces c-fos Expression in Cultured Primary Pituitary Cells. Mol. Endocrinol. 1:300-305 (1987), Billestrup N, et al. Growth Hormone- Releasing Factor Stimulates Proliferation of Somatotrophs In Vitro. Proc. Natl. Acad. Sci USA 83:6854-6857 (1986).
Several observations suggest that the GHRH receptor, in addition to playing a key role in normal growth regulation, might be important in disorders involving aberrant growth hormone secretion. The presumed target for GHRH receptor action in pituitary somatotrophs, the Gsα protein, has been found to be mutated in some growth hormone-secreting pituitary adenomas associated with altered adenylate cyclase activity, Landis C. et al.. GTPase Inhibiting Mutations Activate the α chain of Gs and Stimulate Adenyl Cyclase in Human Pituitary Tumours, Nature 340:692-696 (1989), Vallar L, et al, Altered Gs and Adenylate Cyclase Activity in Human GH-Secreting Pituitary Adenomas. Nature 330:566-568 (1987), suggesting the possibility that the GHRH receptor itself could be a proto-oncogene subject to activation mutations in some pituitary tumors. Cooper JA, Oncogenes and Anti- Oncogenes. Curr. Opin. Cell. Biol. 2:286-295 (1990) . Alterations in the GHRH receptor-Gs-adenylate cyclase system have also been implicated in the growth deficiencies observed in several dwarf rodent strains. Jansson J-O, et al., Receptor-Associated Resistance to Growth Hormone-Releasing Factor in Dwarf "Little" Mice. Science, 232:511-512, Downs T, Frohman L, Evidence for ι Defect in Growth Hormone-Releasing Factor Signal Transduction in the Dwarf (dw/dw) Rat Pituitary. Endocrinology 129:58-67. In particular, the little mutation in the mouse, Eicher E, Beamer J, Inherited Ateliotic Dwarfism in Mice: Characteristics of the Mutation. Little, on Chromosome 6. J. Hered. 67:87-91 (1979) , which is a model for human isolated growth hormone deficiency, involves receptor-associated resistance to GHRH. Jansson J-O, et al. , Receptor- Associated Resistance to Growth Hormone-Releasing Factor in Dwarf "Little" Mice. Science, 232:511-512.
In accordance with the foregoing, potential GHRH receptor cDNA clones were identified from rat and human pituitary. These cDNAs encode proteins that have the expected features of G protein-coupled receptors, and are related to the recently identified secretin and VIP receptors. The human cDNA, when expressed in human kidney 293 cells, produces a protein that binds GHRH with high affinity and specificity and increases GHRH- dependent cAMP production by these transfected cells. The highly related rat cDNA detects an mRNA that is specifically expressed in the rat anterior pituitary gland, the major target for GHRH action.
Summary of the Invention
The present invention provides the cDNA sequence of the pituitary specific receptor for growth hormone releasing hormone as set out in Sequence Id. no. 1 and 2. More specifically, this invention provides a purified human growth hormone-releasing hormone receptor protein selected from the group consisting of a human growth hormone-releasing hormone receptor protein having an amino acid sequence which is at least 85% identical to the amino acid sequence of Sequence Id. No. 1 and fragments thereof comprising at least 10 consecutive amino acids of the sequence. Additionally, this invention provides a purified rat growth hormone- releasing hormone receptor protein selected from the group consisting of a rat growth hormone-releasing hormone receptor protein having an amino acid sequence which is at least 85% identical to the amino acid sequence of Sequence Id. No. 2 and fragments thereof comprising at least 10 consecutive amino acids of the sequence. Additionally, this invention provides a new growth hormone-releasing receptor protein using recombinant DNA molecules capable of expressing this new protein.
Still additionally, this invention provides a method to screen ligands for growth hormone releasing hormone activity. This screening technique could lead to new agonist and antagonist of growth hormone releasing hormone. Description of the Figures
Fig. IA shows the structure of the human pituitary GHRH receptor cDNAs and protein. Schematic maps of human GHRH receptor cDNA clones. A composite cDNA showing the location of the 1269-basepair open reading frame (shaded rectangle) is shown. Above this are the two human cDNA clones isolated, along with a partial restriction map of the HPR3 cDNA.
Fig. IB shows hydropathy plot of the human GHRH receptor protein. Seven strongly hydrophobic regions large enough to span the plasma membrane are numbered 1- 7. Fig. IC shows DNA sequence and amino acid sequence of the human pituitary GHRH receptors. Amino acid 1 corresponds with nucleotides 52-54.
Fig. 2A shows the structure of the rat pituitary GHRH receptor cDNAs and protein. Schematic maps of rat GHRH receptor cDNA clones. A composite cDNA showing the location of the 1269 basepair open reading frame (shaded rectangle) is shown. Above this are the four rat cDNA clones isolated, along with a partial restriction map of the composite cDNA. RPR6 is the initial PCR-generated clone. The triangle indicates an insertion found in clone RPR13 relative to RPR11.
Fig. 2B shows hydropathy plot of the rat GHRH receptor protein. Seven strongly hydrophobic regions large enough to span the plasma membrane are numbered 1- 7.
Fig. 2C shows DNA sequence and amino acid sequence of the rat pituitary GHRH receptor. Amino acid 1 corresponds with nucleotides 28-30. Fig. 3 shows a model for the generation of cDNA clones RPR11 and RPR13 via alternative RNA processing. The upper splicing pattern would generate the RPRll cDNA sequence, while the lower splicing pattern would generate the RPR13 cDNA sequence. Exon sequences are underlined and include amino acid sequence. Note that this model predicts the use of a non-consensus splice donor sequence from the alternative exon (bold characters) .
Fig. 4 shows binding of GHRH by the human GHRH receptor cDNA clone HPR3 in transfected human kidney 293 cells. (A) binding of [l25l-Tyr10] human GHRH (1-44)- amide to membranes from 293-HPR9 cells. The symbols are (•) total binding, (Δ) specific binding, (o) non-specific binding, which was determined by inclusion of excess unlabeled GHRH (1 μM) in parallel samples. Values shown are the means of duplicate samples, which varied by less than 5%. (B) Scatchard analysis of the binding data. Regression analysis was performed using Data Desk 2.0 software. A Kd of 27 pM and 34 pM was determined in two independent experiments, one of which is shown here. (C) Binding competition curve for GHRH in 293-HPR9 cells.
The results of two experiments are shown (• and D) ; each was performed in triplicate and the mean values are shown (values varied by less than 5%) . The EC50 for competition was -50 pM. (D) Specificity of binding of GHRH to 293-HPR9 cells, the amount of input radioligand bound is shown as a function of the cell type (293-WT or 293-HPR9) and the competing peptide, which was present at 1 μM. The dotted line indicates non-specific binding. Measurements were made in quadruplicate and varied by less than 5%.
Fig. 5 shows stimulation of cAMP production by GHRH in human kidney 293 cells expressing the human GHRH receptor. (A) stimulation of cAMP production by GHRH in 293-HPR9 cells. The results from two independent experiments are shown (• and □) ; each was performed in triplicate and the symbols represent the mean plus and minus the standard error. (B) Specificity of cAMP production by 293-HPR9 cells. Relative cAMP levels are shown as a function of the cell type (293-WT or 293-HPR9) and the inducing peptide, which was present at 1 μM. The dotted line shows basal cAMP levels. All measurements were performed" in triplicate and varied by less than 5% with the exception of the GRF induction of HPR9, where values deviated from the mean by 15%. r Fig. 6 shows a schematic diagram of the GHRH receptor as it spans a membrane. It is believed that the amino terminus is on the outside of the cell, while the carboxy terminus is inside of the cell.
Detailed Description of the Invention Novel compositions comprising recombinant proteins produced using generic sequences encoding GHRH receptors and fragments derived therefrom are provided, together with proteins isolated from natural sources, methods of using these compositions. The GHRH receptor cDNAs used to produce the recombinant proteins were initially isolated from human and rat pituitary cDNA libraries using a two-step procedure. First, degenerate oligonucleotides encoding portions of membrane spanning domains 6 and 7 of receptors for calcitonin, Lin H, et al., Expression Cloning of an Adenylate Cvclase-Coupled Calcitonin Receptor. Science 254:1022-1024 (1991), parathyroid hormone, Juppner H, et al, A G-Protein- Linked Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide. Science 254:1024-1026 (1991), and secretin, Ishihara T, Molecular Cloning and
Expression of a cDNA Encoding the Secretin Receptor. EMBO Journal 10:1635-1641 (1991) were used to amplify cDNA generated from the male rat pituitary by the polymerase chain reaction, gel purified, and sequenced. The appropriately sized polymerase chain reaction (PCR) products were cloned and the DNA sequences of several clones were determined.
Standard abbreviations for nucleotides and amino acids are used in these figures and elsewhere in this specification. See Kiefer et aJL. WO 9203471 partially set out to provide general background information. A number of terms used in the art of genetic engineering and protein chemistry are used herein with the following defined meanings. Two nucleic acid fragments are "homologous" if they are capable of hybridizing to one another under hybridization conditions described in Maniatis et al. , op. cit.. pp.320-323. However, by using the following wash conditions — 2 x SCC, 0.1% SDS, room temperature - twice, 30 minutes each; then 2 x SCC, 0.1% SDS, 50°C - once, 30 minutes; then 2 x SCC, room temperature twice, 10 minutes — homologous sequences can be identified that contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15- 25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash conditions for identification of clones from gene libraries (or other sources of genetic material) , as is well known in the art. A DNA fragment is "derived from" a GHRH receptor encoding DNA sequence if it has the same or substantially the same basepair sequence as a region of the coding sequence for the entire GHRH receptor molecule. Substantially the same means, when referring to biological activities, that the activities are of the same type although they may differ in degree. When referring to amino acid sequences, substantially the same means that the molecules in question have similar biological properties and preferably have at least 85% identity in amino acid sequences. More preferably, the amino acid sequences are at least 90% identical. In other uses, substantially the same has its ordinary English language meaning. A protein is "derived from" a GHRH receptor molecule if it has the same or substantially the same amino acid sequence as a region of the GHRH receptor molecule.
GHRH receptor, both glycosylated and unglycosylated, or polypeptide derivatives thereof, may be used for producing antibodies, either monoclonal or polyclonal, specific to GHRH receptors. By polypeptide derivatives of these GHRH receptors is meant polypeptides differing in length from natural GHRH receptor and containing five or more amino acids from GHRH receptor in the same primary order as found in GHRH receptor as obtained from a natural source. Polypeptide molecules having substantially the same amino acid sequence as GHRH receptor but possessing minor amino acid substitutions that do not substantially affect the ability of the GHRH receptor polypeptide derivatives to interact with GHRH receptor-specific molecules. Derivatives include glycosylated forms and covalent conjugates with unrelated chemical moieties.
GHRH receptor-specific molecules include poly- peptides such as antibodies that are specific for the GHRH receptor polypeptide containing the naturally occurring GHRH receptor amino acid sequence. By "specific binding polypeptide" is intended polypeptides that bind with GHRH receptor and its derivatives and which have a measurably higher binding affinity for the target polypeptide, i.e., GHRH receptor and polypeptide derivatives of GHRH receptor, than for other polypeptides tested for binding. Higher affinity by a factor of 10 is preferred, more preferably a factor of 100. Binding affinity for antibodies refers to a single binding event (i.e., monovalent binding of an antibody molecule). Specific binding by antibodies also means that binding takes place at the normal binding site of the molecule's antibody (at the end of the arms in the variable region) . As discussed above, minor amino acid variations from the natural amino acid sequence of GHRH receptor are contemplated as being encompassed by the GHRH receptor; in particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into four families: (a) acidic = aspartate, glutamate; (23) basic = lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding properties of the resulting molecule, especially if the replacement does not involve an amino acid at a binding site involved in the interaction of GHRH receptor. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific binding properties of the GHRH receptor polypeptide derivative.
Antibodies specific for GHRH receptor are produced by immunizing an appropriate vertebrate host, e.g., rabbit, with purified GHRH receptor or polypeptide derivatives of GHRH receptor, by themselves or in conjunction with a conventional adjuvant. Usually, two or more immunizations will be involved, and blood or spleen will be harvested a few days after the last injection. For polyclonal antisera, the immunoglobulins can be precipitated, isolated and purified by a variety of standard techniques, including affinity purification using GHRH receptor attached to a solid surface, such as a gel or beads in an affinity column. For monoclonal antibodies, the splenocytes normally will be fused with an immortalized lymphocyte, e.g., a myeloid cell line, under selective conditions for hybridoma formation. The hybridomas can then be cloned under limiting dilution conditions and their supernatants screened for antibodies having the desired specificity. Techniques for producing antibodies are well known in the literature and are exemplififed by the publication Antibodies: A Laboratory Manual (a988) eds. Harlow and Lane, Cold Spring Harbor Laboratories Press, and U.S. Patent Nos. 4,381,292, 4,451,570, and 4,618,577. GHRH receptor can be readily purified from pituitaries and its components, and from cells genetically modified to produce GHRH receptor or polypeptide derivatives thereof, by affinity chromatography using a monoclonal antibody specific for GHRH receptor. In addition to the use of antibody affinity chromatography, GHRH receptor and polypeptide derivatives thereof can be purified by a variety of other widely known protein purification techniques (either alone or in combination) including immunoprecipitation, gel filtration, ion exchange chromatography, chromato- focusing, isoelectric focusing, selective precipitation, electrophoresis, and the like. Fractions isolated during purification procedures can be analyzed for the presence of GHRH receptor or polypeptide derivatives of GHRH receptor by immunoassays employing GHRH receptor)- specific antibodies or GHRH receptor)-specific bioassays. Detailed examples are provided below.
Isolation of nucleotide sequences encoding GHRH receptor involves creation of either a genomic library or preparation of a cDNA library from RNA isolated from cells expressing GHRH receptor. It will generally be preferable to create a cDNA library for isolation of GHRH receptor coding nucleotide sequences so as to avoid any possible problems arising from attempts to determine intron/exon borders. Genetic libraries can be made in either eukaryotc or prokaryotic host cells. Widely available cloning vectors such as plasmids, cosmids, phage, YACs and the like can be used to generate genetic libraries suitable for the isolation of nucleotide sequences encoding GHRH receptor or portions thereof.
Useful methods for screening genetic libraries for the presence of GHRH receptor nucleotide sequences include the preparation of oligonucleotide probes based on the N-terminus amino acid sequence information from purified GHRH receptor or purified internal fragments of purified GHRH receptor. By employing the standard triplet genetic code, oligonucleotide sequences of about 17 base pairs or longer can be prepared by conventional in vitro synthesis techniques so as to correspond to portions of GHRH receptor for which the amino acid sequence has been determined by N-terminus analysis. The resultant nucleic acid sequences can be subsequently labeled with radionuclides, enzymes, biotin, fluorescers, or the like, and used as probes for screening genetic libraries.
Additional methods of interest for isolating GHRH receptor encoding nucleic acid sequences include screening genetic libraries for the expression of GHRH receptor or fragments thereof by means of GHRH receptor specific antibodies, either polyclonal or monoclonal. A particularly preferred technique involves the use of degenerate primers based on partial amino acid sequences of purified GHRH receptor or on sequences from known related molecules and the polymerase chain reaction (PCR) to amplify gene segments between the primers. The gene can then be isolated using a specific hybridization probe based on the amplified gene segment, which is then analyzed for appropriate expression of protein. A detailed description of this preferred technique is set forth in the examples that follow.
Nucleotide sequences encoding GHRH receptor can be obtained from recombinant DNA molecules recovered from GHRH receptor genetic library isolates. The nucleotide sequence encoding GHRH receptor can be obtained by sequencing the non-vector nucleotide sequences of these recombinant molecules. Nucleotide sequence information can be obtained by employing widely used DNA sequencing protocols, such as Maxim and Gilbert sequencing, dideoxy nucleotide sequencing, and the like. Examples of suitable nucleotide sequencing protocols can be found in Berger and Kimmel, Me€hods in Enzymologv Vol. 52. Guide to Molecular Cloning Techniques, (1987) Academic Press. Nucleotide sequence information from several recombinarr DNA isolates, including isolates from both cDNA and genomic libraries, may be combined so as to provide th»> entire amino acid coding sequence of GHRH receptor, as well as the nucleotide sequences of introns within the GHRH receptor genes, upstream nucleotide sequences, and downstream nucleotide sequences. Nucleotide sequences obtained from sequencing GHRH receptor specific genetic library isolates are subjected to analysis in order to identify regions of interest in the GHRH receptor genes. These regions of interest include open reading frames, introns, promoter sequences, termination sequences, and the like. Analysis of nucleotide sequence information is preferably performed by computer. Software suitable for analyzing nucleotide sequences for regions of interest is commercially available and includes, for example, DNASIS™ (LKB) . It is also of interest to use amino acid sequence information obtained from the N-terminus sequencing of purified GHRH receptor when analyzing GHRH receptor nucleotide sequence information so as to improve the accuracy of the nucleotide sequence analysis. Isolated nucleotide sequences encoding GHRH receptor can be used to produce purified GHRH receptor or fragments thereof by either recombinant DNA methodology or by in vitro polypeptide synthesis techniques. By "purified" and "isolated" is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein preferably means at least 95% by weight, more preferably at least 99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present) . A significant advantage of producing GHRH receptor by recombinant DNA techniques rather than by isolating GHRH receptor from natural sources is that equivalent quantities of GHRH receptor can be produced by using less starting material than would be required for isolating the binding protein from a natural source. Producing. GHRH receptor by recombinant techniques also permits GHRH receptor to be isolated in the absence of some molecules normally present in cells that naturally produce GHRH receptor. Indeed, GHRH receptor compositions entirely free of any trace of human protein contaminants can readily be produced since the only human protein produced by the recombinant nonhuman host is the recombinant GHRH receptor. Potential viral agents from natural sources are also avoided. It is also apparent that recombinant DNA techniques can be used to produce polypeptide derivatives that are not found in nature, such as the variations described above.
GHRH receptor and polypeptide derivatives of GHRH receptor can be expressed by recombinant techniques when a DNA sequence encoding the relevant molecule is functionally inserted into a vector. By "functionally inserted" is meant in proper reading frame and orientation, as is well understood by those skilled in the art. When producing a genetic construction containing a complete GHRH receptor reading frame, the preferred starting material is a cDNA library isolate encoding GHRH receptor than a genomic library isolate. Typically, the GHRH receptor gene will be inserted downstream from a promoter and will be followed by a stop codon, although production as a hybrid protein followed by cleavage may be used, if desired. In general, host- cell-specific sequences improving the production yield of GHRH receptor and GHRH receptor polypeptide derivatives will be used and appropriate control sequences will be added to the expression vector, such as enhancer sequences, polyadenylation sequences, and ribosome binding sites.
Once the appropriate coding sequence is isolated, it can be expressed in a variety of different expression systems, or it can be inserted into the genome for transgenic expression. Mammalian Expression Systems
A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, typically located within 100 to 200 bp upstream of the TATA box.
An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation (Sambrook et ai. (1989) Expression of Cloned Genes in Mammalian Cells," in Molecular Cloning: A Laboratory Manual. 2nd ed.) .
Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP) , and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible) , and depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.
The presence of an enhancer element (enhancer) , combined with the promoter elements described above, will typically increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from t he promoter (Maniatis et al.. (1989) Molecular Biology of the Cell. 2nd ed.). Enhancer elements derived from viruses may be particularly useful, because they typically have a broader host range. Examples include the SV40 early gene enhancer (Dijkema et al. (1985) EMBO J. 4:761) and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gormal et aJ (1982b) Proc. Natl. Acad. Sci. 79:6777) and from human cytomegalovirus (Boshart et al. (1985) Cell 41:521). Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion (Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237).
A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide. Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3 • to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation (Birnstiel et al. (1985) Cell 41;349; Proudfoot and Whitelaw (1988) "Termination And 3' end processing of eukaryotic RNA." In Transcription and splicing (ed. B.D. Hames and D.M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105). These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator/polyadenylation signals include those derived from SV40 (Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells." In Molecular Cloning; A Laboratory Manual) .
Some genes may be expressed more efficiently when introns (also called intervening sequences) are present Several cDNAs, however, have been efficiently expresse ι from vectors that lack splicing signals (also called spliced donor and acceptor sites) (see e.g., Gothing and Sambrook (1981) Nature 293:620). Introns are intervening noncoding sequences within a coding sequence that contain spliced donor and acceptor sites. They are removed by a process called "splicing" following polyadenylation of the primary transcript (Nevins (1983) Annu. Rev. Biochem. 52:441; Green (1986) Annu. Rev. Genet. 20:671; Padgett et al. (1986) Annu. Rev. Biochem. 55:1119; Krainer and Maniatis (1988) "RNA splicing." In Transcription and splicing (ed. B.D. Hames and D.M. Glover)).
Typically, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 (Gluzman (1981) Cell 23:175) or polyomavirus, replicate to extremely high copy number in the presence of the T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a procaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 (Kaufman et a_A_ (1989) Mol. Cell. Biol. 9:946 and pHEBO (Shimizu et al^ (1986) Mol. Cell. Biol. 6:1074). Alternatively, foreign proteins can also be targeted to the membrane of a mammalian cell. If the cDNA expression construct includes an amino-terminal hydrophobic leader sequence, and one or more additional internal hydrophobic domains of sufficient size to span the cell membrane (typically -20 amino acids) , the resulting protein can be targeted to the cell membrane and retained there in a conformation dependent on the nature and characteristics of the internal hydrophobic domains. (Wickner W.T. and Lodish H.F., Multiple Mechanisms of Protein Insertion into and Across Membranes. Science 300:400-407 (1985)). (Hereby incorporated by reference) .
Baculovirus Expression System
A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (3*) transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5* end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A baculovirus promoter may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.
Sequences encoding genes abundantly transcribed at late times in the infection cycle provide particularly useful promoter sequences. Examples include sequences derived from the polyhedrin (Friesen et al. (1986) "The Regulation of Baculovirus Gene Expression," in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler) ; E.P.O. Pub. Nos. 127,839 and 155,476) and plO (Vlak et al. (1988) J. Gen. Virol. 69:765) genes. A DNA molecular may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which the case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
Fusion proteins provide an alternative to direct expression. Typically, a DNA sequence encoding the N- terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the N-terminus of the polyhedrin gene may be linked at the 5' terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See e.g., Luckow et al. (1988) Bio/technology 6:47.
Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene
(Carbonell et al. (1988) Gene 73:409). Alternatively, leaders of non-baculovirus origin, such as those derived from genes encoding human alpha-interferon (Maeda et al. (1985) Nature 315:592), human gastrin-releasing peptide (Lebacq-Verheyden et al. (1988) Molec. Cell. Biol. 8:3129), human IL-2 (Smith et a . (1985) Proc. Natl. Acad. Sci. USA 82:8404), mouse IL-3 (Miyajima et al. (1987) Gene 58:273), and human glucocerebrosidase (Martin et al. (1988) DNA 7:99) also provide for secretion in insects.
Typically, transcription termination sequences recognized by insects are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples include transcription termination sequences derived from the polyhedrin gene (Miller et al. (1988) Ann. Rev. Microbiol. 42:177). Prior to insertion of the foreign gene into the baculovirus genome, the above described components, comprising a promoter, leader (if desired) , coding sequence of interest, and transcription termination sequence, are typically put together into an intermediate transplacement construct. Intermediate transplacement constructions are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host for cloning and amplification. The promoter and transcription termination sequence of the construct will typically comprise a 2.5kb section of the baculovirus genome for integration of the foreign gene into the baculovirus genome by double crossover recombination events, producing a baculovirus expression vector (Miller et al. (1989) Bioessays 4:91). The baculovirus expression vector is typically packaged into an infectious recombinant baculovirus. When using baculovirus expression vectors, selectable markers are, such as antibiotic resistance genes, are generally not used. Selection is typically by visual inspection for occlusion bodies. Examples are given elsewhere in this specification of the use of selectable markers.
Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for inter alia: Aβdes aegypti, A tographa californica, Bombyx mori, Drosophila melanogaster, Heliothis zea, Spodoptera frugiperda, and Tric oplusiain (P.C.T. WO 89/046699; Carbonell et aj^ (1985) J. Virol. 56:153: Smith et al*. (1983) Mol. Cell. Biol. 3:2156; Wright (1986) Nature 321:718; See generally, Fraser et S-L. (1989) In Vjtfp ς*U . pgvτ Bjς>;t 25:225).
Methods of introducing exogenous DNA into insect hosts are well-known in the art, and typically include either the transfection of host insect cells with DNA or the infection of insect cells or live insects, usually larvae, with virus. Transfection procedures are based on the calcium phosphate procedure originally developed for mammalian cells (Graham ej£ al. (1973) Virology 52:456) . DNA transfection and viral infection procedures usually vary with the insect genus to be transformed. See e.g. Autograph (Carstens et al. (1980) Virology 101:311), Heliothis (virescens) (P.C.T. Pub. No. W088/02030) , Spodoptera (Kang (1988) "Baculovirus Vectors for Expression of Foreign Genes," in Advances in Virus Research, vol. 35).
Alternatively, foreign proteins can also be targeted to the membrane of an insect cell. If the cDNA expression construct includes an amino-terminal hydrophobic leader sequence, and one or more additiona. internal hydrophobic domains of sufficient size to spa.-. the cell membrane (typically -20 amino acids) , the resulting protein can be targeted to the cell membrane and retained there in a conformation dependent on the nature and characteristics of the internal hydrophobic domains. (Wickner W.T. and Lodish H.F., Multiple Mechanisms of Protein Insertion into and Across Membranes. Science 300:400-407 (1985)). (Hereby incorporated by reference) .
Bacterial Expression Systems
A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g., structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA * polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP) , which helps initiate transcription of the lac operon in Escherichia coli (E. coli) (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Example include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1977) Nature 198:1056). and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al*. (1981) Nucl. Acids Res. 9:731: U.S. Patent No. 4,738,921; E.P.O. Pub. Nos. 36,776 and 121,775). The γ- lactamase (bla) promoter system (Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3. (ed. I. Gresser), bacteriophage lambda PL (Shimatake et al. (1981) Nature 292:128) and T5 (U.S. Patent No. 4,689,406) promoter systems also provide useful promoter sequences.
In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter (U.S. Patent No. 4,551,433). For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor (Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21). Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to product high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1985) Proc. Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E_j_ coli operator region (E.P.O. Pub. No. 267,851) .
In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In JL. coli. the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (Shine et al. (1975) Nature 254:34). The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3 • and of E . coli 16S rRNA (Steitz et al. (1979) "Genetic signals and nucleotide sequences in messenger RNA." In Biological Regulation and Development: Gene Expression (ed. R.F. Goldberger) ) . To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site (Sambrook et al. (1989) Expression of Cloned genes in Escherichia coli." In Molecular Cloning; A Laboratory Manual) .
A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N- terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N- terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo or in vitro incubation with a bacterial methionine N- terminal peptidase (E.P.O. Pub. No. 219,237). Fusion proteins provide an alternative to direct expression. Typically, a DNA sequence encoding the N- terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5' terminus of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave, the bacteriophage protein from the foreign gene (Nagai et al. (1984) Nature 309:810). Fusion proteins can also be made with sequences from the lacZ (Jia et aJ . (1987) Gene 60:197), troE (Allen et aJ (1987) J. Biotechnol. 5:93; Makoff et aA. (1989) J. Gen. Microbiol. 135:11), and Chev (E.P.O. Pub. No. 324,647) genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated (Miller et al. 91989) Bio/Technology 7:698).
Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria (U.S. Patent No.
4,336,336). The signal sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell(gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E_j. coli outer membrane protein gene (ompA) (Masui et al. (1983) , in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212). As an additional example, the signal sequence of the alpha- amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B___ subtilis (Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; E.P.O. Pub. No. 244,042) .
Typically, transcription termination sequences recognized by bacteria are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forminq stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E^. coli as well as other biosynthetic genes.
Typically, the above described components, comprising a promoter, signal sequence (if desired) , coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintains ι in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a procaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either. a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and typically about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors typically contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (E.P.O. Pub. No. 127,328). Integrating vectors may also be comprised of bacteriophage or transposon sequences. Typically, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin) , and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469). Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are typically comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis (Palva et al.. (1982) Proc. Natl. Acad. Sci. USA 79:5582; E.P.O. Pub. Nos. 36,259 and 63,953; P.C.T. WO 84/04541), Escherichia coli (Shimatake et al. (1981) Nature 292:128; Aman et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; E.P.O. Pub. Nos. 36,776, 136,829 and 136,907; U.K. Patent Application Serial No. 8418273), Streptococcus cremoris (Powell et al. (1988) Appl. Environ. Microbiol. 54:655); Streptococcus lividans (Powell et al. (1988) Appl. Environ. Microbiol. 54:655), Streptomyces lividans (U.S. Patent No. 4,745,056).
Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and typically include either the transformation of bacteria treated with CaCl2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See e.g. , (Masson et a . (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; E.P.O. Pub. Nos. 36,259 and 63,953; P.C.T. WO 84/04541, Bacillus) , (Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter) , (Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) "An improved method for transformation of Escherichia coli with EolEl-derived plasmids. In Genetic Engineering; Proceedings of the International Symposium on Genetic Engineering (eds. H.W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia) (Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus) ; (Fiedler et al. (1988) Anal.
Biochem. 170:38, Pseudomonas) ; (Augustin et al. (1990) FEMS Microbiol. Lett 66:203, Staphylococcus) , (Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) "Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J.
Ferretti and R. Curtiss III) ; Perry et al. (1981) Infec. Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Eyr. Cong. Biotechnology 1:412, Streptococcus . Alternatively, foreign proteins can also be targeted to the membrane of a bacterial cell. If the cDNA expression construct includes an amino-terminal hydrophobic leader sequence, and one or more additional internal hydrophobic domains of sufficient size to span the cell membrane (typically -20 amino acids) , the resulting protein can be targeted to the cell membrane and retained there in a conformation dependent on the nature and characteristics of the internal hydrophobic domains. (Wickner W.T. and Lodish H.F., Multiple Mechanisms of Protein Insertion into and Across Membranes. Science 300:400-407 (1985)). (Hereby incorporated by reference) .
Description: Yeast Expression System
A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA. A yeast expression system is particularly important in the present application for the expression of the binding protein which is expected to be extracellular. See Fig. 6. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site (the "TATA Box") and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS) , which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.
Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydorgenase (ADH) (E.P.O. Pub No. 284044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH) , hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (E.P.O. Pub. No. 329203) . The yeast PHQ5 gene, encoding acid phosphatase, also provides useful promoter sequences (Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1). In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Patent Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2. GAL4. GAL10. or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (E.P.O. Pub No. 164556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yease RNA polymerase and initiate transcription. Examples of such promoters include inter alia. (Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al^. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96: 119; Hollenberg et al. (1979) "The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae," in Plasmids of Medical, Environmental and Commercial Importance (eds. K.N. Timmis and A. Puhler; Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109).
A DNA molecle may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant progein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
Fusion proteins provide an alternative to direct expression. Typically, a DNA sequence encoding the N- terminal portion of an' endogenous yeast protein, or oth. r stable protein, is fused to the 5* end of heterologous coding sequences. Upon expression, this construct wil. provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD, gene, can be linked at the 5' terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See e.g., E.P.O. Pub. No. 196056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene tht can be cleaved either in vivo or in vitro. The leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (E.P.O. Pub. No. 13873; J.P.O. Pub. No. 62,096,086) and the A-factor gene (U.S. Patent No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (E.P.O. Pub. No. 60057).
A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre" signal sequence, and a "pro" region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (typically about 25 to about 50 amino acid residues) (U.S. Patent Nos. 4,546,083 and 4,870,008; E.P.O. Pub. No. 324274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast slphafactor. (See e.g., P.C.T. WO 89/02463) .
Typically, transcription termination sequences recognized by yeast are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.
Typically, the above described components, comprising a promoter, leader (if desired) , coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained ina replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a procaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 (Botstein et al. (1979) Gene 8:17- 24), pCl/1 (Brake et al^_ (1984) Proc Natl. Acad. Sci. USA 81:4642-4646), and YRpl7 (Stinchcomb et al. (1982) J . Mol. Biol. 158:157). In addition, a replicon may be either a h igh or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and typically about 10 to about 150. A host containing a high copy number plasmit will preferably have at least about 10, and more preferably at least about 20. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See e.g. Brake et al. , supra. Alternatively, the expression constructs can be integrated into the yeast genome with an integrating . vector. Integrating vectors typically contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome (Orr-Weaver et al. (1983) Methods in Enzymol. 101:228- 245) . An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al. , supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced (Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750). The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.
Typically, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2. HIS4. LEU2. TRP1. and ALG7. and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions (Butt et al. (1987) Microbiol. Rev. 51:351). Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are typically comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans
(Kurtz, et a . (1986) Mol. Cell. Biol. 6:142), Candida maltosa (Kunze, et al. (1985) J. Basic Microbiol. 25:141). Hansenula polymorpha (Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302), Kluyveromyces fragilis (Das, et al. (1984) J. Bacteriol. 158:1165), Kluyveromyces lact is (De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135), Pichia guillerimondii (Kunze et al. (1985) J. Basic Microbiol. 25:141), Pichia pastoris (Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Patent Nos. 4,827,148 and 4,929,555), Saccharomyces cerevisiae (Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al^ (1983) J Bacteriol. 153:163) Schizosaccharomyces pombe (Beach and Nurse (1981) Nature 300:706), and Yarrowia lipolytica (Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49).
Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and typically include either the transformation of spheroplasts or of intact 41)
yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See e.g., (Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze ejt al. (1985) J. Basic Microbiol. 25:141; Candida ) ; (Gleeson et al. (1986) J. Gen.
Microbiol. 132:3459; Roggenka p et al. (1986) Mol. Gen. Genet. 202:302; Hansenula ; (Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces) ; (Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et aL.(1985) Is. Basic Microbiol. 25:141; U.S. Patent Nos. 4,837,48 and 4,929,555; Pichia ; (Hinnen et als. (1978) Proc. Natl. Acad. Sc. USA 75:1929; Ito et aA_ (1983) J. Bacteriol. 153:163 Saccharomyces) ; (Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces) ; (Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia . Alternatively, foreign proteins can also be targeted to the membrane of a yeast cell. If the cDNA expression construct includes an amino-terminal hydrophobic leader sequence, and one or more additional internal hydrophobic domains of sufficient size to spar, the cell membrane (typically -20 amino acids) , the resulting protein can be targeted to the cell membrane and retained there in a conformation dependent on the nature and characteristics of the internal hydrophobic domains. (Wickner W.T. and Lodish H.F., Multiple Mechanisms of Protein Insertion into and Across Membranes. Science 300:400-407 (1985)). (Hereby incorporated by reference) .
Diagnostic Applications using Genetic Probes
The genetic material of the invention can itself . used in numerous assays as probes for genetic ateria. present in naturally occurring materials. The analytt- can be a nucleotide sequence which hybridizes with a probe comprising a sequence of (usually) at least about 16 consecutive nucleotides, usually 30 to 200 nucleotides, up to substantially the full sequence of the sequences shown above (cDNA sequences) . The analyte can be RNA or cDNA. The sample is typically as described in the previous section. A positive result is generally characterized as identifying a genetic material comprising a sequence at least about 70% identical to a sequence of at least 12 consecutive nucleotides of the sequences given herein, usually at least about 80% homologous to at least about 60 consecutive nucleotides within the sequences, and may comprise a sequence substantially identical to the full-length sequences. In order to detect an analyte, where the analyte hybridizes to a probe, the probe may contain a detectable label. Probes that are particularly useful for detecting RNA or DNA for receptor proteins are based on conserved regions of these proteins, particularly from amino acids 131 and amino acids 380 (See Fig. 6) showing the membrane spanning domain.
One method for amplification of target nucleic acids, for later analysis by hybridization assays, is known as the polymerase chain reaction or PCR technique. The PCR technique can be applied to detecting GHRH receptor of the invention in suspected samples using oligonucleotide primers spaced apart from each other and based on the genetic sequence set forth herein. The primers are complementary to opposite strands of a double stranded DNA molecule and are typically separated by from about 50 to 450 nt or more (usually not more than 2000 nt) . This method entails preparing the specific oligonucleotide primers and then repeated cycles of target DNA denaturation, primer binding, and extension with a DNA polymerase to obtain DNA fragments of the expected length based on the primer spacing. Extension products generated from one primer serve as additional target sequences for the other primer. The degree of amplification of a target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2n where n is the number of cycles. Given that the average efficiency per cycle ranges from about 65% to 85%, 25 cycles produce from 0.3 to 4.8 million copies of the target sequence. The PCR method is described in a number of publications, including Saiki et al.. Science (1985) 2309:1350-1354; Saiki et al.. Nature (1986) 324:163-166; and Scharf et al.. Science (1986) 233:1076-1078. Also see U.S. Patent Nos. 4,683,194; 4,683,195; and 4,683,202. The invention includes a specific diagnostic method for determination of GHRH receptor, based on selective amplification of GHRH receptor-encoding RNA fragments from tissue. This method employs using reverse transcriptase to make a cDNA sequence and then amplifying this sequence with receptor specific primers. These "primer fragments," which form one aspect of the invention, are prepared from GHRH receptor fragments such as described above. The method follows the process for amplifying selected nucleic acid sequences as disclosed in U.S. Patent No. 4,683,202, as discussed above.
Uses of GHRH Receptors
The present cDNA sequences provide proteins that can be used to screen altered ligands as agonists or antagonists to growth hormone releasing hormone. These altered ligands could be used to treat diseases associated with growth hormone releasing hormone. Similarly, with the cDNA sequence known, a protein could be designed to bind to growth hormone releasing hormone. This new receptor administered in a pharmaceutically acceptable carrier may be used to control growth rates in domestic animals. Additionally, human growth disorders can be assessed using either an antibody to the GHRH receptor or a nucleic acid probe to the receptor.
Isolation and Analysis of Receptor cDNA Clones
The approach used to identify candidate GHRH receptor clones was based on the amplification of G protein-coupled receptor cDNAs using degenerate oligonucleotide primers, Libert F. , et al. Selective Amplification and Cloning of Four New Members of the G Protein-Coupled Receptor Family. Science 244:569-572 (1989) . Primers were designed based on the sequences of a family of structurally related receptors, including receptors for calcitonin, Lin H., et al.. Expression Cloning of an Adenylate Cyclase-Coupled Calcitonin Receptor. Science 254:1022-1024 (1991), parathyroid hormone, Juppner H, et al. A G-Protein-Linked Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide. Science 254:1024-1026 (1991), and secretin,
Ishihara T., et al. Molecular Cloning and Expression of a cDNA encoding the secretin receptor. EMBO Journal 10:1635-1641 (1991). The relatedness between the secretin and GHRH ligands suggested that their receptors might also be homologous, and the recent finding that the calcitonin, PTH, and secretin receptors form a sub-family of G protein-coupled receptors, Lin H. , et al.. Expression Cloning of an Adenylate Cvclase-Coupled Calcitonin Receptor. Science 254:1022-1024 (1991), Juppner H, et al. A G-Protein-Linked Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide. Science 254:1024-1026 (1991), and secretin, Ishihara T., et al. Molecular Cloning and Expression of a cDNA encoding the secretin receptor. EMBO Journal 10:1635-1641 (1991) provided a structural basis for designing primers that might amplify additional family members. Degenerate oligonucleotides encoding portions of membrane spanning domains 6 and 7 of these receptors were used to amplify cDNA generated from male rat pituitary. The appropriately sized polymerase chain reaction (PCR) products were cloned, and the DNA sequences of several of the clones was determined.
One of the clones isolated, RPR6, encoded a peptide with strong homology to the targeted regions of the calcitonin, PTH and secretin receptors. RPR6 was used to screen human and rat pituitary cDNA libraries, and a human cDNA clone of 1617 bases (HPR3) was identified that contained an open reading frame encoding a 47 kilodalton protein of 423 amino acids (Figura IA and Sequence Id. No. 1) . Subsequent screening of the rat pituitary library identified 3 clones (RPR64, RPR11, and RPR20) that together span 1629 bases and encode a 47 kilodalton protein of 423 amino acids that is 82% identical to the human protein (Figure 2C and Sequence Id. No. 2) . HPR3 and RPRC (the composite rat cDNA) both encode proteins with seven hydrophobic potential membrane spanning domains (Figures IB and 2B) , suggesting that they might be G protein-coupled receptors. As expected based on the cloning strategy, the predicted protein product of RPRC is related to the somewhat larger calcitonin and PTH receptors (24% and 27% amino acid identity, respectively) . Even greater sequence identity was found between RPRC and the rat secretin receptor (35% identity) and the recently identified rat vasoactive intestinal peptide (VIP) receptor (40% identity) ,
Ishihara T, et al.. Functional Expression and Tissue Distribution of a Novel Receptor for Vasoactive Intestinal Peptide. Neuron 8:811-819 (1992). Similar relatedness between the predicted protein product of HPR3, the human cDNA, and the calcitonin, PTH, secretin, and VIP receptors was found. No additional strong nucleic acid or protein homologies were found in searches of the GenBank and Swiss-Prot databases.
One of the rat cDNA clones isolated (RPR13) was found to contain an additional 123 bases in the coding sequence compared to another rat cDNA (RPR11) or to the human cDNA. this is predicted to insert 41 amino acids into the encoded protein immediately before membrane- spanning domain 6; the sequence of this insert is shown in Sequence Id. No. 3. See Fig. 3. Analysis of rat genomic clones indicates that this insertion point corresponds to an intron-exon boundary, suggesting that alternative RNA processing generates the larger receptor isoform. the presumed splicing patterns by which these alternate products are generated are shown in Figure 3A. Preliminary analysis of rat pituitary mRNA using reverse transcription-polymerase chain reaction and primers that span the insertion site identified only the shorter form of the transcript; however, it is possible that expression of these mRNA isoforms in the pituitary is hormonally or developmentally regulated.
Expression of the GHRH Receptor in Transfected Cells
The structural relatedness between the RPRC and HPR3 receptors and the rat secretin and VIP receptors suggested that the ligand for this novel receptor would indeed be a member of the family of peptides that includes secretin, VIP, GHRH, GIP, PHI, PACAP and glucagon, Rivier J, et al.. Characterization of a Growth Hormone-Releasing Factor from a Human Pancreatic Islet Tumour. Nature 300:276-278 (1982), Bell GI, The Glucagcn Superfamily: Precursor Structure and Gene Organization. Peptides 7 (Suppl) :27-36 (1986), Miyata A., et al.. Isolation of a Novel 38 Residue Hvpothalamic Polypept . Which Stimulates Adenylate Cyclase in Pituitary Cells . Biochem Biophys. Res. Comm 164:567-574 (1989). To examine the ligand binding characteristics of this putative receptor, the HPR3 cDNA was cloned into the mammalian cell expression vector pcDNA-1 and was used to generate stable lines of human kidney 293 cells by cotransfection with a G418 resistance selectable marker, pSV2-neo. A clonal line (293-HPR9) that had several copies of the transfected gene and expressed high amounts of receptor mRNA was selected for further analysis. Crude membrane fractions from these cells, or from the control 293 cells (293-WT) , were used for binding studies with the ligand (125I-Tyr10) human GHRH 91-44) -amide, Abribat T. , et al . Characterization of (125i-Tyrl£) Human Growth Hormone Releasing Factor (1-44) Amide Binding to Rat Pituitary: Evidence for High and Low Affinity
Classes of Sites. Brain Research 528:291-299 (1990) . As shown in Figure 4A, hGHRH bound to membranes from the transfected cell line 293-HRP9 in a saturable fashion. Scatchard analysis of the binding data, shown in Figure 4B, revealed a dissociation constant for hGHRH interaction with the receptor in 293-HPR9 cells of 30 pM with approximately 50,000 binding sites per cell. As such, in screening for ligands as agonists of GHRH, an affinity of about 30 pM is expected. GHRH binding to the receptor in 293-HPR9 cells was also examined in competition experiments, which are shown in Figure 4C. The EC50 for competition of radioligand binding by hGHRH was found to be -50 pM. These values are in reasonable agreement witht he reported dissociation constants for binding of hGHRH (1-44) -amide or the related agonist (His1, Nle27) hGHRH (1-32) -amide to rat pituitary membranes, which range from 41 pM to 680 pM, Abribat T. , et al. Characterization of ( Hi-Tyr ^) Human Growth Hormone Releasing Factor (1-44) Amide Binding to Rat Pituitary: Evidence for High and Low Affinity Classes of Sites. Brain Research 528:291-299 (1990), Seifert H, et al. Binding Sites for Growth Hormone Releasing Factor on Rat Anterior Pituitary Cells. Nature 313:487-489 (1985), Aribrat T, et al. Alterations of Pituitrv Growth Hormone- Releasing factor Binding in Aging Rats. Endocrinology 128:633-635 (1991), Seifert H, et al. Growth Hormone- Releasing Factor Binding Sites in Rat Anterior Pituitary Membrane Homogenates: Modulation by Glucocorticoids. Endocrinology 117:424 (1985). The binding specificity was examined in competition experiments, which are shown in Figure 4D. No specific binding to control 293-WT cells was observed, and the binding to 293-HPR9 cells could be completely competed to 293-WT levels by the addition of excess unlabeled hGHRH (1-44) -amide (Figure 4D) or the commonly used GHRH agonist (His1, Nle27) hGHR (1-32)-amide. Secretin did not compete binding of the GHRH radioligand at all, and VIP competed binding of the GHRH radioligand only weakly (EC50>lμM) . These results demonstrate that the protein encoded by HPR3 is a high affinity GHRH receptor.
To determine whether GHRH binding to 293 cells transfected with the cloned GHRH receptor results in the activation of adenylate cyclase, cells were stimulated with GHRH, secretin, or VIP and intracellular cAMP levels were measured by radioimmunoassay. Increasing doses of GHRH resulted in elevated cAMP levels in the 293-HPR9 cells (Figure 5A) . The EC50 for cAMP accumulation was approximately 5nM, which is substantially higher thatn the EC50 determined in binding-competition experiments. There are several possible explanations for this, including desensitization of the receptor int he presence of IBMX and high ligand concentrations, inefficient coupling of the over-expressed receptor to downstream components of the signal transduction system in this cell line, and degradation of the ligand by cell or serum- associated proteases in the absence of protease inhibitors, which were present in the in vitro binding expreiments, Struthers R, et al. , Nucleotide Regulation of Growth Hormone-Releasing Factor Binding to Rat Pituitary Receptors. Endocrinology 124:24-29 (1989). The specificity of GHRH action on these transfected cells is demonstrated in Figure 5. GHRH treatment resulted in a large increase in cAMP levels in 293-HPR9 cells, but had very little effect on cAMP levels in the control 293-WT cells. Secretin had no effect on cAMP levels in the transfected 293-HPR9 cells, but VIP did increase cAMP to about 5% the level obtained with GHRH (Figure 5B) , consistent with the ability of VIP to bind to the receptor with low affinity. The activation of the intracellular signaling system upon the binding of a ligand to GHRH is shown by increased production of cyclic AMP or protein kinase C. Expression of GHRH Receptor mRNA in Pituitary
The tissue distribution of GHRH receptor mRNA expression was examined in the rat using the RPR64 cDNA as a hybridization probe. A predominant transcript of -2.5 kilobases, as well as a less abundant transcript of -4 kilobases, are observed specifically in the pituitary lane. Additionally, it was observed that all lanes of the RNA blot contained roughly equivalent amounts of RNA. The pituitary expression of the rat GHRH receptor was also analyzed by in situ hybridization using 35s- labeled RNA probes. GHRH mRNA was not detected in the liver, but was detected in the pituitary using an antisense RNA probe. No hybridization was observed when a control sense-strand GHRH receptor probe was used. The GHRH receptor mRNA co-localized with the mRNA for the pituitary-specific transcription factor Pit-1, Bodner M, et al. The Pituitary-Specific Transcription Factor GHF-1 is a Homeobox-Containing Protein.. Cell 55:505-518 (1988) , Ingraham, et al.. A Tissue-Specific Transcription Factor Containing a Homeodomain Specifies a Pituitary Phenotype, Cell 55:519-529 (1988), and was completely restricted to the anterior lobe of the pituitary gland.
Example 1 - Isoaltion and Analysis of GHRH Receptor Clones
Degenerate oligonucleotide primers to membrane spanning domains 6 (5'-ACCCTC[CGA]TNCTG[GA]T[CG] CCGCTC[TC]T[TC]GG-3* (Sequence Id. No. 4) and 7 (5'-
TGCAC[CT]TCA[CT] [CG] [AG]TTG[AC] [AG] [AG]AA[AG]CA[AG]TA-3 ' (Sequence Id. No. 5) of the secretin, calcitonin, and PTH receptors were used to amplify cDNA generated from male rat pituitaries. N is G, A, T or C. Amplification was carried out for 30 cycles using an annealing temperature of 55°C on a Perkin Elmer Cetus thermocycler and Taq DNA polymerase (Perkin Elmer Cetus, Norwalt, CT) . The product was gel-purified and cloned into pGEM3Z (Promega, Madison, WI) for DNA sequence analysis. One of the clones isolated, RPR6 was subsequently used to screen human and rat cDNA libraries, which were from Clonetech (Palo Alto, CA) . The human pituitary library was constructed from a growth hormone-producing adenoma from a female patient in λbluemid, the rat pituitary library was constructed from adult males in λgtlO. Screening and plaque purification was performed using standard methods, and inserts were subcloned into pGEM3Z or pGEM7Z (Promega, Madison, WI) for ruther analysis. cDNA clones were sequenced on both strands using modified T7 polymerase (United States Biochemicals, Cleveland OH) and dideoxynucleotides. DNA sequence manipulations were performed using GeneWorks 2.1 software from Intelligenetics (Mountain View, CA) , Hydrophobicity was analyzed using the algorithm of Kyte and Doolittle, Kyte and Doolittle, A Simple Model for Displaying the Hydropathic Character of a Protein. J. Mol. Biol. 157:105-132 (1982), with an analysis window of 11 amino acids. Amino acid sequence alignment was performed using GeneWorks 2.1 software and was subsequently manually modified to maximize alignment of the cysteine residues.
Cell Transfection. Binding Assays, and cAMP Measurements
Human cDNA clone HPR3 was subcloned into the eukaryotic expression vector pcDNA-1 (Invitrogen, San Diego, CA) using lipofectin reagent (Gibco-BRL,
Gaithersburg, MD) , and a 9:1 μg per 100 mm plate ratio of the HPR3 cDNA expression construct to pSV2neo, Southern P and Berg P, Transformation of Mammalian Cells to Antibiotic Resistance with a Bacterial Gene Under the Control of the SV40 Early Region Promoter. J. Mol. Appl. Genet. 1:327-341 (1982). Transfected cells were selected in 400 μg/ml G418 (Sigma Chemical, St. Louis, MI) and individual clones wee isolated and expanded for subsequent analysis. Clone HPR9 was selected for further analysis based on high-level expression of the cDNA, and was maintained continuously in 400 μg/ml G418. For binding assays, control 293-WT cells or transfected 293- HPR9 cells were washed with phosphate-buffered saline and homogenized (20 strokes in a TEFLON-glass unit) on ice in 50 mM Tris-HCl pH 7.4, 5mM MgCL2, 2 mM EGTA, and 0.1 πM PMSF. The homogenate was centrifuged for 5 minutes at lOOXg to remove larger material, and the supernatant was re-centrifuged for 10 minutes at 4000Xg. The membrane pellet was resuspended in binding buffer: 25 mM Hepes LH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.1 mM PMSF, o . mg/ml leupeptin, 1 mg/ml bacitractin and 0.1% bovine serum albumin. Binding reactions were performed at 23 ' for 60 minutes in a volume of 0.5 ml with -50 μg me br ■■ protein. For competition experiments, (125l-Tyr10) - - GHRH (1-44) -amide (Amersham, Arlington Heights, IL) * present at -75 pM. Binding reactions were terminated by centrifugation in a microfuge for 5 minutes in the cold. The supernatant was aspirated, and the tip of the tube containing the pellet was cut off and counted in a gamma counter. Total binding was generally 30-45% of input, while non-specific binding was 8-11% of input.
For cAMP determinations, cells were grown in 6-well plates, and were treated with 0.1 mM IBMX for 20 minutes at 37°C. Hormones were added in fresh warmed media, and the incubation was continued for another 20 minutes at 37°C. Medium was removed, 0.3 ml cold C.l N HC1 was added to each well, and cell lysates were stored at -20°C until cAMP radioimmunoassays were performed as described, Nikaido S and Takahashi J, Twenty-four Hour Oscillations of cAMP in Chick Pineal Cells: Role of cAMP in the Acute and Circadian Regulation of Melatonin Production. Neuron 3:609-619 (1989). All peptides were the human sequences, and they were obtained from Peptides International (Louisville, KY) or Peninsula Laboratories (Belmont, CA) .
RNA Blot Analysis and in Situ Hybridization
RNA was prepared from the indicated tissues by homgenization in guanidine isothiocynate and centrifugation through cesium chloride, and polyadenylated RNA was selected by chromatography using oligo(dT) cellulose. Approximately 10 μg of each RNA was separated by electrophoresis on denaturing 1% agarose/formaldehyde gels. RNA was transferred to a nylon membrane (ICN, Irvine, CA) , covalently attached by UV cross-linking, and detected by hybridization to the insert from RPR-64 that had been labeled with 32P-dCTP using random hexamer primers and the Klenow fragment of E. coli DNA polymerase. Hybridization was performed in 50% formamide, 5X SSPE, 2X Denhardt's reagent, 10% dextran sulfate, 0.1% SDS, and 100 μg/ml salmon sperm DNA. The membranes were subsequently washed in 0.1X SSC at 65°C and exposed to Kodak XAR-5 film. After removal of probe in 50% formamide at 65°C, the membranes were re- hybidized to cDNA clone CHO-B, Harpold MM, et al. Production of mRNA in Chinese Hamster Cells:
Relationship of the Rate of Synthesis to the Cytoplasmic Concentration of Nine Specific mRNA Sequences. Cell 17:1025-1035 (1979), which detects the LLRep3 gene family. Heller DL, et al. A Highly Conserved Mouse Gene With a Propensity to Form Pseudooenes in Mammals. Mol. Cell. Biol. 8:2797-2803 (1988), to assess the amount of RNA present in each lane.
For in situ hybridization, frozen tissues were removed from storage at -80°C and brought to -20°C, and 20 μm sections were cut using a Reichert 820 cryostat (Buffalo, NY) . Sections were mounted onto gelatin- and poly L-lysine-coated glass slides for processing as described previously, Suhr ST, et al." Mouse Growth Hormone-Releasing Hormone: Precursor Structure and Expression in Brain and Placenta. Mol. Endocrinol. 3:1693-1700 (1989). In brief, ovarian sections were fixed in 5% paraformaldehyde (pH 7.5) for 5 min, washed in 2X SSC followed by 0.1 M triethanolamine (pH 8.0) and incubated in 0.25% acetic anhydride in 0.1 M tri- ethanolamine (pH 8.0) for 10 min. Sections were dehydrated using ethanol and vacuum dried. Antiserse and sense (35S)UTP-labeled RNA probes were synthesized using T7 or SP6 polymerase. The RNA probe (2X107 cpm/ml in hybridization buffer: 50% formamide, 5X SSPE, 2X Denhardt's reagent, 10% dextran sulfate, 0.1% SDS, and
100 μg/ml yeast tRNA) was applied and the tissue sections were overlaid with a coverslip. Slides were hybridized in a humidity chamber at 47°C for 16-18 hours. After hybridization, the coverslips were removed and sections were treated with RNase A (20 μg/ml) at 37°C for 30 min, washed in increasingly lower concentrations of SSC down to 0.1X SSC at 55°C and dehydrated using ethanol. The slides were exposed to betamax film (Amersham, Arlington Heights, IL) for 2-e days and were then processed for liquid emulsion autoradiography sing NTB-2 emulsion (Kodak Company, Rochester, NY) .
It should be understood that this invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiment is therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing discussion, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
54 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: MAYO, KELLY E.
(ii) TITLE OF INVENTION: NEW GROWTH HORMONE RELEASING HORMONE
RECEPTOR PROTEIN
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
AGCAGCCAAG GCTTACTGAG GCTGGTGGAG GGAGCCACTG CTGGGCTCAC CATGGACCGC 60 CGGATGTGGG GGGCCCACGT CTTCTGCGTG TTGAGCCCGT TACCGACCGT ATTGGGCCAC 120
ATGCACCCAG AATGTGACTT CATCACCCAG CTGAGAGAGG ATGAGAGTGC CTGTCTACAA 180
GCAGCAGAGG AGATGCCCAA CACCACCCTG GGCTGCCCTG CGACCTGGGA TGGGCTGCTG 240
TGCTGGCCAA CGGCAGGCTC TGGCGAGTGG GTCACCCTCC CCTGCCCGGA TTTCTTCTCT 300
CACTTCAGCT CAGAGTCAGG GGCTGTGAAA CGGGATTGTA CTATCACTGG CTGGTCTGAG 360 CCCTTTCCAC CTTACCCTGT GGCCTGCCCT GTGCCTCTGG AGCTGCTGGC TGAGGAGGAA 420
TCTTACTTCT CCACAGTGAA GATTATCTAC ACCGTGGGCC ATAGCATCTC TATTGTAGCC 480
CTCTTCGTGG CCATCACCAT CCTGGTTGCT CTCAGGAGGC TCCACTGCCC CCGGAACTAC 540
GTCCACACCC AGCTGTTCAC CACTTTTATC CTCAAGGCGG GACGTGTGTT CCTGAAGGAT 600
GCTGCCCTTT TCCACAGCGA CGACACTGAC CACTGCAGCT TCTCCACTGT TCTATGCAAG 660 GTCTCTGTGG CCGCCTCCCA TTTCGCCACC ATGACCAACT TCAGCTGGCT GTTGGCAGAA "20
GCCGTCTACC TGAACTGCCT CCTGGCCTCC ACCTCCCCCA GCTCAAGGAG AGCCTTCTGG "30
TGGCTGGTTC TCGCTGGCTG GGGGCTGCCC GTGCTCTTCA CTGGCACGTG GGTGAGCTGC =40
AAACTGGCCT TCGAGGACAT CGCGTGCTGG GACCTGGACG ACACCTCCCC CTACTGGTGG -:0
ATCATCAAAG GGCCCATTGT CCTCTCGGTC GGGGTGAACT TTGGGCTTTT TCTCAATATT - - ATCCGCATCC TGGTGAGGAA ACTGGAGCCA GCTCAGGGCA GCCTCCATAC CCAGTCTCAG .. ■.
TATTGGCGTC TCTCCAAGTC GACACTTTTC CTGATCCCAC TCTTTGGAAT TCACTACATC . -
ATCTTCAACT TCCTGCCAGA CAATGCTGGC CTGGGCATCC GCCTCCCCCT GGAGCTGGGA . . - 3
CTGGGTTCCT TCCAGGGCTT CATTGTTGCC ATCCTCTACT GCTTCCTCAA CCAAGAGGTG .. .
AGGACTGAGA TCTCACGGAA GTGGCATGGC CATGACCCTG AGCTTCTGCC AGCCTGGAGG \ : - 1 ACCCGTGCTA AGTGGACCAC GCCTTCCCGC TCGGCGGCAA AGGTGCTGAC ATCTATGTGC . .
TAGGCTGCCT CATCACGCCA CTGGAGTCCA CACTTGAATT TGGGCAGCTA CCACGGGTCT .
GCCATGCTCT GGAGGAGCAA GGGGGCCACA TCCCCACCCC AGCTGTTACC CAGCCCGGCG
CAGGTGCAGC CCTTCCTCCC TGTCTCTGCA TCTGACTCTC TTTTGAGGTC CCTGTATGT TACCTCTGAC TTCTGTGGTC CCTCTGTGTC TGCTCTCATC CATTCCTCTT ACTGGGGCCT 1560
GGGGCTCTAG CCCAAGGCTC AGAGGAGCCA ATAAACCTGT AAATGAAAAA AAAAAAA 1617
(2) INFORMATION FOR SEQ ID NO:2: ^
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1629 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOG : unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AAACAGCGGA CTACAGGCAC CACTGCCATG GACAGCCTGT TGTGGGCTAC CTGGGTCCTC 60 TGCTTGCTGA ACCTGTGGGG AGTTGCACTG GGTCACCTCC ACCTAGAATG TGACTTCATC 120
ACTCAGCTGA GAGACGATGA GCTTGCATGC CTTCAGGCGG CAGAGGGGAC CAACAACTCG 180
TCCATGGGAT GCCCTGGGAC CTGGGATGGG CTGCTGTGCT GGCCCCCAAC TGGCTCTGGC 240
CAGTGGGTCT CCCTCCCCTG CCCTGAATTC TTCTCTCATT TTGGCTCAGA CCCAGGGGCT 300
GTGAAAAGGG ACTGCACCAT CACGGGTTGG TCTGATCCCT TCCCACCATA TCCCGTGGCC 360 TGTCCTGTGC CCTTGGAACT GCTAACAGAG GAGAAGTCTT ACTTCTCCAC GGTGAAGATC 420
ATCTACACCA CAGGCCACAG CATCTCCATT GTAGCCCTCT GCGTGGCTAT TGCCATCCTG 480
GTTGCTCTCA GGAGGCTCCA CTGCCCCAGG AACTACATCC ACACGCAGCT GTTTGCTACT 540
TTCATCCTCA AGGCCAGTGC TGTGTTCCTG AAGGATGCTG CTGTCTTCCA GGGTGATAGC 600
ACGGACCACT GCAGCATGTC CACTATTCTG TGCAAGGTCT CTGTGGCCGT CTCACATTTT 660 GCCACCATGA CCAACTTCAG CTGGCTGCTG GCAGAAGCCG TCTACCTGAG CTGTCTGTTG 720
GCCTCCACAT CTCCTAGGTC CAAACCAGCT TTCTGGTGGC TGGTTCTCGC TGGCTGGGGA 780
CTCCCTGTGC TATGCACTGG TACGTGGGTG GGCTGCAAAC TGGCTTTTGA GGACACTGCG 840
TGCTGGGACC TAGACGACAG CTCCCCCTAC TGGTGGATCA TCAAAGGGCC CATAGTCCTC 900
TCTGTTGGGG TGAACTTTGG GCTGTTTCTC AATATAATTT GCATCCTGCT GAGGAAGCTG 960 GGGCCTGCAC AAGGCGGCTT ACACACACGG GCTCAGTACT GGCGGCTTTC CAAATCAACA 1020
CTTCTCCTTA TCCCGCTGTT TGGAATTCAT TACATCATCT TCAACTTCCT GCCTGACAGT 1080
GCTGGCCTTG GCATCCGTCT ACCCCTGGAG CTGGGACTGG GGTCCTTCCA GGGTTTTGTT 1140
GTTGCTGTCC TCTACTGCTT CCTCAATCAA GAGGTGAGGA CGGAGATTTC ACGCAAATGG 1200
TATGGCCATG ACCCTGAACT TCTGCCAGCT CGGCGGACCT GCACTGAGTG GACCACACCT 1260 CCCCGATCGA GAGTGAAGGT GCTCACCTCT GAGTGCTAGG CCAGCCATCA CAAAGGCCGA 1320
GCCCCAAAAC CCTGCACTCA AACTGCCATG CCACCAAGGG CAACAAGGTC CTCCCTTCCG 1380 TTCTCATTCT CTGCATCTGC TTTCTCTAGG TCCCTGTATA CCAACCTCCG ACTTTCTCAG 1440
TTCCTGTATG CCCCCATCTG TTCTTTCTTC CTATCTAGGC TATTGCCCAA GGCCCAGGGA 1500
AACCAATAAA CTTGTACATG AGTGATCTGC AGTTGAGTCA ATGTGGCTCT GAAGGGGAGC 1560
TCTTGTCAGC AGCCATTATT TGCACTTCCG GTGCATTCCT CATCCCTTGG CTGCAGCTGC 1620 CTCATTGCC .1629
(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CAACTATTTA TTACCCTGGT CCTGTCCTCT TCCTCAAGTT CCTAGAGAGA GAACAGATCT 60
GGGCCCTTCG TCCCATGAGA TTACAGTACA GGAAAGTGGC ACACGAAACT GCCAGTTACC 120
ATG 123
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ACCCTCCGAT NCTGGATCGC CGCTCTCTTC GG 32
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TGCACCTTCA CTCGACTTGA CAGAGAAAGC AAG 33

Claims

I CLAIM:
1. A purified human growth hormone-releasing hormone receptor protein selected from the group consisting of a human growth hormone-releasing hormone receptor protein having an amino acid sequence which is at least 85% identical to the amino acid sequence of Sequence Id. No. 1 and fragments thereof comprising at least 10 consecutive amino acids of said sequence. 2. The protein of Claim 1 wherein said protein comprises the amino acid sequence of Sequence Id. No. 1. 3. The protein of Claim 1 wherein said protein comprises one of said fragments. 4. Recombinant human growth hormone-releasing hormone receptor protein. 5. An antibody, antibody fragment, or derivative thereof which recognizes human growth hormone- releasing hormone receptor protein. 6. A purified rat growth hormone-releasing hormone receptor protein selected from the group consisting of a rat growth hormone-releasing hormone receptor protein having an amino acid sequence which is at least 85% identical to the amino acid sequence of Sequence Id. No. 2 and fragments thereof comprising at least 10 consecutive amino acids of said sequence.
7. The protein of Claim 5 wherein said protein comprises the amino acid sequence of Sequence Id. No. 2.
8. The protein of Claim 5 wherein said protein comprises one of said fragments.
9. Recombinant rat growth hormone-releasing hormone receptor protein. 10. A purified human growth hormone releasing hormone receptor binding protein selected from the group consisting of a human growth hormone-releasing hormone receptor binding protein having an amino acid sequence which is at least 85% identical to the amino acid sequence of Sequence Id. No. 1 from about amino acids 1 to 130 of Sequence Id. No. 1 and fragments thereof comprising at least 10 consecutive amino acids of said sequence. ii. Recombinant human growth hormone-releasing hormone receptor, binding protein.
12. A purified rat growth hormone releasing hormone receptor binding protein selected from the group consisting of a rat growth hormone-releasing hormone receptor binding protein having an amino acid sequence which is at least 85% identical to the amino acid sequence of Sequence Id. No. 2 from about amino acids 1 to 130 of Sequence Id. No. 2 and fragments thereof comprising at least 10 consecutive amino acids of said sequence.
13. Recombinant rat growth hormone-'releasing hormone receptor binding protein.
14. A method to screen ligands for human growth hormone- releasing hormone activity comprising: (a) contacting cells, expressing said human growth hormone-releasing hormone receptor protein of claims 4 or 9 with a ligand; (b) detecting whether said human growth hormone- releasing hormone binds said ligand; and (c) detecting activation of the intracellular signalling system in said contacted cells.
15. The method of claim 14 wherein said cells are human kidney 293 cells.
16. The method of claim 14 wherein said cells are present in a transgenic animal. 17. The method of claim 14 wherein said cells are yeast cells.
18. The method of claim 14 wherein said intracellular signal is protein kinase C. 19. The method of claim 14 wherein said intracellular signal is cyclic AMP.
20. Essentially purified oligonucleotide probes for identifying growth hormone releasing hormone receptor consisting of nucleotides selected from Sequence Id. No. 1.
21. The oligonucleotide probes of claim 20 wherein said nucleotides code for amino acids in the membrane spanning domain.
22. Essentially purified oligonucleotide probes for identifying growth hormone releasing hormone receptor consisting of nucleotides selected from Sequence Id. No. 2.
23. The oligonucleotide probes of claim 22 wherein said nucleotides code for amino acids in the membrane spanning domain.
PCT/US1993/009084 1992-09-28 1993-09-23 New growth hormone releasing hormone receptor protein WO1994007915A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US95349392A 1992-09-28 1992-09-28
US07/953,493 1992-09-28

Publications (1)

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WO1994007915A1 true WO1994007915A1 (en) 1994-04-14

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU673188B2 (en) * 1992-09-15 1996-10-31 Bruce David Gaylinn Isolation, characterization, and protein sequencing of the growth hormone releasing hormone receptor and cloning of a gene encoding for the growth hormone releasing hormone receptor
US5852187A (en) * 1992-06-23 1998-12-22 The University Of Virginia Patent Foundation Molecular cloning of the ovine pituitary growth hormone releasing hormone receptor
WO2000009538A2 (en) * 1998-08-10 2000-02-24 Merck & Co., Inc. Canine growth hormone secretagogue receptor

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
EMBO J., Volume 10(7), issued 1991, T. ISHIHARA et al., "Molecular Cloning and Expression of a cDNA Encoding the Secretin Receptor", pages 1635-1641. *
J. BIOLOGICAL CHEMISTRY, Volume 261(36), issued 25 December 1986, J.R. ZYSK et al., "Cross-Linking of a Growth Hormone Releasing Factor-Binding Protein in Anterior Pituitary Cells", pages 16781-16784. *
MOLECULAR ENDOCRINOLOGY, Volume 6(10), issued 1992, K. MAYO, "Molecular Cloning and Expression of a Pituitary-Specific Receptor for Growth Hormone-Releasing Hormone", pages 1734-1744. *
PEPTIDES, Volume 13, issued 1992, R.M. CAMPBELL et al., "Synthesis and Biological Activity of Novel C-Terminal-Extended and Biotinylated Growth Hormone Factor (GRF) Analogs", pages 787-793. *
SCIENCE, Volume 254, issued 15 November 1991, J. JUPPNER et al., "A G Protein-Linked Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide", pages 1024-1026. *
SCIENCE, Volume 54, issued 15 November 1991, H.Y. LIN et al., "Expression Cloning of an Adenylate Cyclase-Coupled Calcitonin Receptor", pages 1022-1024. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5852187A (en) * 1992-06-23 1998-12-22 The University Of Virginia Patent Foundation Molecular cloning of the ovine pituitary growth hormone releasing hormone receptor
AU673188B2 (en) * 1992-09-15 1996-10-31 Bruce David Gaylinn Isolation, characterization, and protein sequencing of the growth hormone releasing hormone receptor and cloning of a gene encoding for the growth hormone releasing hormone receptor
WO2000009538A2 (en) * 1998-08-10 2000-02-24 Merck & Co., Inc. Canine growth hormone secretagogue receptor
WO2000009538A3 (en) * 1998-08-10 2000-06-22 Merck & Co Inc Canine growth hormone secretagogue receptor
US6645726B1 (en) 1998-08-10 2003-11-11 Merck & Co., Inc. Canine growth hormone secretagogue receptor

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